Fad4, fad5, fad5-2, and fad6, novel fatty acid desaturase family members and uses thereof

ABSTRACT

The invention provides isolated nucleic acid molecules which encode novel fatty acid desaturase family members. The invention also provides recombinant expression vectors containing desaturase nucleic acid molecules, host cells into which the expression vectors have been introduced, and methods for large-scale production of long chain polyunsaturated fatty acids (LCPUFAs), e.g., DHA.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/173,799, filed Jun. 6, 2016, which is a continuation of U.S.application Ser. No. 13/339,428, filed Dec. 29, 2011, now U.S. Pat. No.9,359,597, which is a divisional of U.S. application Ser. No.13/150,656, filed Jun. 1, 2011, now U.S. Pat. No. 8,088,906, which is adivisional of U.S. application Ser. No. 12/538,227, filed Aug. 10, 2009,now U.S. Pat. No. 7,977,469, which is a divisional of U.S. applicationSer. No. 11/342,731, filed Jan. 30, 2006, now U.S. Pat. No. 7,671,252,which is a divisional of U.S. application Ser. No. 09/967,477, filedSep. 28, 2001, now now U.S. Pat. No. 7,087,432, which claims priority toU.S. Provisional Application No. 60/236,303 filed on Sep. 28, 2000 andU.S. Provisional Application No. 60/297,562 filed on Jun. 12, 2001. Theentire contents of each of these applications are hereby incorporated byreference herein in their entirety. The entire contents of allreferences cited therein also are expressly incorporated by referenceand are intended to be part of the present application.

SUBMISSION OF SEQUENCE LISTING

The Sequence Listing associated with this application is filed inelectronic format via EFS-Web and hereby incorporated by reference intothe specification in its entirety. The name of the text file containingthe Sequence Listing is 074008_1323_02_ST25. The size of the text fileis 45,706 bytes, and the text file was created on Aug. 1, 2019.

BACKGROUND OF THE INVENTION

Fatty acids are carboxylic acids with long-chain hydrocarbon side groupsand play a fundamental role in many biological processes. Fatty acidsare rarely free in nature but, rather, occur in esterified form as themajor component of lipids. Lipids/fatty acids are sources of energy(e.g., b-oxidation) and are an integral part of cell membranes which areindispensable for processing biological or biochemical information.

Fatty acids can be divided into two groups: the saturated fatty acidsand the unsaturated fatty acids which contain one or more carbon doublebond in cis-configuration. Unsaturated fatty acids are produced byterminal desaturases that belong to the class of nonheme-iron enzymes.Each of these enzymes are part of a electron-transport system thatcontains two other proteins, namely cytochrome b5 and NADH-cytochrome b5reductase. Specifically, such enzymes catalyze the formation of doublebonds between the carbon atoms of a fatty acid molecule. Human and othermammals have a limited spectrum of these desaturases that are requiredfor the formation of particular double bonds in unsaturated fatty acids.Thus, humans have to take up some fatty acids through their diet. Suchessential fatty acids, for example, are linoleic acid (C18:2); linolenicacid (C18:3), arachidonic acid (C20:4). In contrast, insects and plantsare able to synthesize a much larger variety of unsaturated fatty acidsand their derivatives.

Long chain polyunsaturated fatty acids (LCPUFAs) such as docosahexaenoicacid (DHA, 22:6(4,7,10,13,16,19)) are essential components of cellmembranes of various tissues and organelles in mammals (nerve, retina,brain and immune cells). For example, over 30% of fatty acids in brainphospholipid are 22:6 (n−3) and 20:4 (n−6). (Crawford, M. A., et al.,(1997) Am. J. Clin. Nutr. 66:1032S-1041S). In retina, DHA accounts formore than 60% of the total fatty acids in the rod outer segment, thephotosensitive part of the photoreceptor cell. (Giusto, N. M., et al.(2000) Prog. Lipid Res. 39:315-391). Clinical studies have shown thatDHA is essential for the growth and development of the brain in infants,and for maintenance of normal brain function in adults (Martinetz, M.(1992) J. Pediatr. 120:S129-S138). DHA also has significant effects onphotoreceptor function involved in the signal transduction process,rhodopsin activation, and rod and cone development (Giusto, N. M., etal. (2000) Prog. Lipid Res. 39:315-391). In addition, some positiveeffects of DHA were also found on diseases such as hypertension,arthritis, atherosclerosis, depression, thrombosis and cancers(Horrocks, L. A. and Yeo, Y. K. (1999) Pharmacol. Res. 40:211-215).Therefore, the appropriate dietary supply of the fatty acid is importantfor humans to remain healthy. It is particularly important for infant,young children and senior citizens to adequately intake these fattyacids from the diet since they cannot be efficiently synthesized intheir body and must be supplemented by food (Spector, A. A. (1999)Lipids 34:S1-S3).

DHA is a fatty acid of the n−3 series according to the location of thelast double bond in the methyl end. It is synthesized via alternatingsteps of desaturation and elongation. Starting with 18:3 (9,12,15),biosynthesis of DHA involves Δ6 desaturation to 18:4 (6,9,12,15),followed by elongation to 20:4 (8,11,14,17) and Δ5 desaturation to 20:5(5,8,11,14,17). Beyond this point, there are some controversies aboutthe biosynthesis. The conventional view is that 20:5 (5,8,11,14,17) iselongated to 22:5 (7,10,13,16,19) and then converted to 22:6(4,7,10,13,16,19) by the final Δ4 desaturation (Horrobin, D. F. (1992)Prog. Lipid Res. 31:163-194). However, Sprecher et al. recentlysuggested an alternative pathway for DHA biosynthesis, which isindependent of Δ4 desaturase, involving two consecutive elongations, aΔ6 desaturation and a two-carbon shortening via limited β-oxidation inperoxisome (Sprecher, H., et al. (1995) J. Lipid Res. 36:2471-2477;Sprecher, H., et al. (1999) Lipids 34:S153-S156).

Production of DHA is important because of its beneficial effect on humanhealth. Currently the major sources of DHA are oils from fish and algae.Fish oil is a major and traditional source for this fatty acid, however,it is usually oxidized by the time it is sold. In addition, the supplyof the oil is highly variable and its source is in jeopardy with theshrinking fish populations while the algal source is expensive due tolow yield and the high costs of extraction.

EPA and AA are both Δ5 essential fatty acids. They form a unique classof food and feed constituents for humans and animals. EPA belongs to then−3 series with five double bonds in the acyl chain, is found in marinefood, and is abundant in oily fish from North Atlantic. AA belongs tothe n−6 series with four double bonds. The lack of a double bond in thew-3 position confers on AA different properties than those found in EPA.The eicosanoids produced from AA have strong inflammatory and plateletaggregating properties, whereas those derived from EPA haveanti-inflammatory and anti-platelet aggregating properties. AA can beobtained from some foods such as meat, fish, and eggs, but theconcentration is low.

Gamma-linolenic acid (GLA) is another essential fatty acid found inmammals. GLA is the metabolic intermediate for very long chain n−6 fattyacids and for various active molecules. In mammals, formation of longchain polyunsaturated fatty acids is rate-limited by Δ6 desaturation.Many physiological and pathological conditions such as aging, stress,diabetes, eczema, and some infections have been shown to depress the Δ6desaturation step. In addition, GLA is readily catabolized from theoxidation and rapid cell division associated with certain disorders,e.g., cancer or inflammation. Therefore, dietary supplementation withGLA can reduce the risks of these disorders. Clinical studies have shownthat dietary supplementation with GLA is effective in treating somepathological conditions such as atopic eczema, premenstrual syndrome,diabetes, hypercholesterolemia, and inflammatory and cardiovasculardisorders.

The predominant sources of GLA are oils from plants such as eveningprimrose (Oenothera biennis), borage (Borago officinalis L.), blackcurrant (Ribes nigrum), and from microorganisms such as Mortierella sp.,Mucor sp., and Cyanobacteria. However, these GLA sources are not idealfor dietary supplementation due to large fluctuations in availabilityand costs associated with extraction processes.

SUMMARY OF THE INVENTION

The biosynthesis of fatty acids is a major activity of plants andmicroorganisms. However, humans have a limited capacity for synthesizingessential fatty acids, e.g., long chain polyunsaturated fatty acids(LCPUFAs). Biotechnology has long been considered an efficient way tomanipulate the process of producing fatty acids in plants andmicroorganisms. It is cost-effective and renewable with little sideeffects. Thus, tremendous industrial effort directed to the productionof various compounds including speciality fatty acids and pharmaceuticalpolypeptides through the manipulation of plant, animal, andmicroorganismal cells has ensued. Accordingly, biotechnology is anattractive route for producing unsaturated fatty acids, especiallyLCPUFAs, in a safe, cost-efficient manner so as to garner the maximumtherapeutic value from these fatty acids.

The present invention is based, at least in part, on the discovery of afamily of nucleic acid molecules encoding novel desaturases. Inparticular, the present inventors have identified the Fad 4 (Δ4desaturase), Fad5 and Fad5-2 (Δ5 desaturase), and Fad6 (Δ6 desaturase)which are involved in the biosynthesis of long chain polyunsaturatedfatty acids DHA (docosahexaenoic acid, 22:6, n−3) and DPA(docosapentaenoic acid, 22:5, n−6); more specifically, Fad4 desaturases22:5 (n−3) and 22:4 (n−6) resulting in DHA and DPA; Fad5 and Fad5-2desaturases 20:4 (n−3) and 20:3(n−6) resulting in EPA and AA; and Fad6desaturases 18:2 (n−6) and 18:3(n−3) resulting in GLA (gamma-linolenicacid) and SDA (stearidonic acid).

In one embodiment, the invention features an isolated nucleic acidmolecule that includes the nucleotide sequence set forth in SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7. In another embodiment, theinvention features an isolated nucleic acid molecule that encodes apolypeptide including the amino acid sequence set forth in SEQ ID NO:2,4, 6, or 8.

In still other embodiments, the invention features isolated nucleic acidmolecules including nucleotide sequences that are substantiallyidentical (e.g., 70% identical) to the nucleotide sequence set forth asSEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7. The inventionfurther features isolated nucleic acid molecules including at least 30contiguous nucleotides of the nucleotide sequence set forth as SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7. In another embodiment,the invention features isolated nucleic acid molecules which encode apolypeptide including an amino acid sequence that is substantiallyidentical (e.g., 50% identical) to the amino acid sequence set forth asSEQ ID NO:2, 4, 6, or 8. Also featured are nucleic acid molecules whichencode allelic variants of the polypeptide having the amino acidsequence set forth as SEQ ID NO: 2, 4, 6, or 8. In addition to isolatednucleic acid molecules encoding full-length polypeptides, the presentinvention also features nucleic acid molecules which encode fragments,for example, biologically active fragments, of the full-lengthpolypeptides of the present invention (e.g., fragments including atleast 10 contiguous amino acid residues of the amino acid sequence ofSEQ ID NO: 2, 4, 6, or 8). In still other embodiments, the inventionfeatures nucleic acid molecules that are complementary to, or hybridizeunder stringent conditions to the isolated nucleic acid moleculesdescribed herein.

In a related aspect, the invention provides vectors including theisolated nucleic acid molecules described herein (e.g.,desaturase-encoding nucleic acid molecules). Also featured are hostcells including such vectors (e.g., host cells including vectorssuitable for producing desaturase nucleic acid molecules andpolypeptides).

In another aspect, the invention features isolated desaturasepolypeptides and/or biologically active fragments thereof. Exemplaryembodiments feature a polypeptide including the amino acid sequence setforth as SEQ ID NO: 2, 4, 6, or 8, a polypeptide including an amino acidsequence at least 50% identical to the amino acid sequence set forth asSEQ ID NO: 2, 4, 6, or 8, a polypeptide encoded by a nucleic acidmolecule including a nucleotide sequence at least 70% identical to thenucleotide sequence set forth as SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5,or SEQ ID NO:7. Also featured are fragments of the full-lengthpolypeptides described herein (e.g., fragments including at least 10contiguous amino acid residues of the sequence set forth as SEQ ID NO:2, 4, 6, or 8) as well as allelic variants of the polypeptide having theamino acid sequence set forth as SEQ ID NO: 2, 4, 6, or 8.

In one embodiment, a desaturase polypeptide or fragment thereof has adesaturase activity. In another embodiment, a desaturase polypeptide, orfragment thereof, has an N-terminal heme-binding motif, e.g., acytochrome b5-like domain found in front-end desaturases. In anotherembodiment, a desaturase polypeptide, or fragment thereof, has at leasttwo, preferably about three, conservative histidine motifs found in allmicrosomal desaturases and, optionally, has a desaturase activity. In apreferred embodiment, the desaturase polypeptide, or fragment thereof,has about three histidine motifs.

The constructs containing the desaturase genes can be used in anyexpression system including plants, animals, and microorganisms for theproduction of cells capable of producing LCPUFAs such as DHA, EPA, AA,SDA, and GLA. Examples of plants used for expressing the desaturases ofthe present invention include, among others, plants and plant seeds fromoilseed crops, e.g., flax (Linum sp.), rapeseed (Brassica sp.), soybean(Glycine and Soja sp.), sunflower (Helianthus sp.), corron (Gossypiumsp.), corn (Zea mays), olive (Olea sp.), safflower (Carthamus sp.),cocoa (Theobroma cacoa), and peanut (Arachis sp.).

In a related aspect, the present invention provides new and improvedmethods of producing unsaturated fatty acids, e.g., LCPUFAs, and otherkey compounds of the unsaturated fatty acid biosynthetic pathway usingcells, e.g., plant cells, animal cells, and/or microbial cells in whichthe unsaturated fatty acid biosynthetic pathway has been manipulatedsuch that LCPUFAs or other desired unsaturated fatty acid compounds areproduced.

The new and improved methodologies of the present invention includemethods of producing unsaturated fatty acids (e.g., DHA) in cells havingat least one fatty acid desaturase of the unsaturated fatty acidbiosynthetic pathway manipulated such that unsaturated fatty acids areproduced (e.g., produced at an increased level). For example, theinvention features methods of producing an unsaturated fatty acid (e.g.,DHA) in cells comprising at least one isolated desaturase nucleic acidmolecule, e.g., Fad4, Fad5, Fad5-2, and/or Fad6, or a portion thereof,as described above, such that an unsaturated fatty acid, e.g., LCPUFA,e.g., DHA, is produced. Such methods can further comprise the step ofrecovering the LCPUFA.

In another embodiment, the present invention provides methods ofproducing unsaturated fatty acids, e.g., LCPUFAs, e.g., DHA, comprisingcontacting a composition comprising at least one desaturase targetmolecule, as defined herein, with at least one isolated desaturasepolypeptide, e.g., Fad4, Fad5, Fad5-2, and/or Fad6, or a portionthereof, as described above, under conditions such that an unsaturatedfatty acid, e.g., LCPUFA, e.g., DHA, is produced. Such methods canfurther comprise the step of recovering the LCPUFA.

The nucleic acids, proteins, and vectors described above areparticularly useful in the methodologies of the present invention. Inparticular, the invention features methods of enhancing unsaturatedfatty acid production (e.g., DHA production) that include culturing arecombinant plant, animal, and/or microorganism comprising a desaturasenucleic acid, e.g., Fad4, Fad5, Fad5-2, and/or Fad6, under conditionssuch that fatty acid production is enhanced.

In another embodiment, the present invention features methods ofproducing a cell capable of producing unsaturated fatty acids. Suchmethods include introducing into a cell, e.g., a plant cell, an isolatednucleic acid molecule which encodes a protein having an activity ofcatalyzing the formation of a double bond in a fatty acid molecule.

In another embodiment, the present invention features methods formodulating the production of fatty acids comprising culturing a cellcomprising an isolated nucleic acid molecule which encodes a polypeptidehaving an activity of catalyzing the formation of a double bond, suchthat modulation of fatty acid production occurs.

In another embodiment, the present invention includes compositions whichcomprise the unsaturated fatty acids nucleic acids or polypeptidesdescribed herein. Compositions of the present invention can alsocomprise the cells capable of producing such fatty acids, as describedabove, and, optionally, a pharmaceutically acceptable carrier.

In another embodiment, the compositions of the present invention areused as a dietary supplement, e.g., in animal feed or as aneutraceutical. The compositions of the present invention are also usedto treat a patient having a disorder, comprising administering thecomposition such that the patient is treated. Disorders encompassed bysuch methods include, for example, stress, diabetes, cancer,inflammatory disorders, and cardiovascular disorders.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show the DNA and protein sequence of Fad4 fromThraustochytrium sp.; FIG. 1A shows the cDNA sequence of the openreading frame (SEQ ID NO:1); and FIG. 1B shows the translated proteinsequence (SEQ ID NO:2).

FIGS. 2A and 2B show the DNA and protein sequence of Fad5 fromThraustochytrium sp.; FIG. 2A shows the cDNA sequence of the openreading frame (SEQ ID NO:3); and FIG. 2B shows the translated proteinsequence (SEQ ID NO:4).

FIG. 3 shows a comparison of Fad4 and Fad5 protein sequences fromThraustochytrium sp. (SEQ ID NO:2 and 4, respectively). The vertical barindicates amino acid identity. The conserved motifs such as thecytochrome b5 heme-binding and the histidine-rich motifs arehighlighted. The two arrows indicate the binding locations of the twodegenerate primers.

FIGS. 4A and 4B show the DNA and protein sequence of Fad5-2 from Pythiumirregulare; FIG. 4A shows the cDNA sequence of the open reading frame(SEQ ID NO:5); and FIG. 4B shows the translated protein sequence (SEQ IDNO:6).

FIGS. 5A and 5B show the DNA and protein sequence of Fad6 of Pythiumirregulare; FIG. 5A shows the cDNA sequence of the open reading frame(SEQ ID NO:7); and FIG. 5B shows the translated protein sequence (SEQ IDNO:8).

FIG. 6 shows a comparison of Fad5-2 and Fad6 protein sequences fromPythium irregulare (SEQ ID NO: 6 and 8, respectively). The vertical barindicates amino acid identity. The conserved motifs such as thecytochrome b5 heme-binding and the histidine-rich motifs arehighlighted. The two arrows indicate the binding locations of the twodegenerate primers.

FIG. 7 is a gas chromatographic (GC) analysis of fatty acid methylesters (FAMEs) from yeast strain Invsc2 expressing Fad4 with exogenoussubstrate 22:5 (n−3).

FIGS. 8A and 8B are a gas chromatographic/mass spectroscopy (MS)analysis of FAMEs of the new peak in FIG. 7; FIG. 8A shows the Fad4product; FIG. 8B shows the DHA (22:6, n−3) standard.

FIG. 9 is a GC analysis of FAMEs from yeast strain Invsc2 expressingFad4 with exogenous substrate 22:4 (n−6).

FIGS. 10A and 10B are a GC/MS analysis FAMEs of the new peak in FIG. 9;FIG. 10A shows the Fad4 product; FIG. 10B shows the DPA (22:5, n−6)standard.

FIG. 11 is a GC analysis of FAMEs from yeast strain Invsc2 expressingFad5 with exogenous substrate 20:3 (n−6).

FIGS. 12A and 12B are a GC/MS analysis of FAMES of the new peak in FIG.11; FIG. 12A shows the Fad5 product; FIG. 12B shows the AA(20:4-5,8,11,14) standard.

FIG. 13 is a GC analysis of FAMES from yeast strain AMY2α expressingFad5-2 with exogenous substrate 18:1-9 (the upper panes) and 18:1-11(the lower panel), respectively.

FIG. 14 is a GC analysis of FAMEs from yeast strain Invsc2 expressingFad6 with exogenous substrate 18:2 (9,12).

FIG. 15 is a MS analysis of the derivative of the new peak from FIG. 14.The structure of the diethylamide of the new fatty acid is shown withm/z values for ions that include the amide moiety. The three pairs ofions at m/z, 156/168, 196/208, and 236/248 are diagnostic for doublebonds at the Δ6, Δ9, and Δ12 position, respectively.

FIG. 16 is a GC analysis of FAMEs from leaves of Brassica junceaexpressing Fad4 under the control of 35S promoter with exogenouslysupplied substrate 22:5 (n−3).

FIG. 17 shows the fatty acid composition of vegetative tissues (leaves,stems, and roots) of one transgenic T1 line with Fad5-2 under thecontrol of the 35S promoter. The fatty acid levels are shown as theweight percentage of total fatty acids in B. juncea.

FIG. 18 is a GC analysis of root FAMEs of B. juncea expressing Fad5-2with exogenous substrate homo-γ-linolenic acid (HGLA, 20:3-8,11,14).

FIG. 19 is a GC analysis of FAMEs prepared from seeds of B. junceaexpressing Fad5-2 under the control of the napin promoter.

FIG. 20 is a GC analysis of seed FAMEs from B. juncea expressing Fad6.Three new peaks indicate three Δ6 desaturated fatty acids in transgenicseeds.

FIG. 21 shows the weight percentage of GLA (γ-linolenic acid) and SDA(stearidonic acid) accumulating in Fad6 transgenic seeds of B. juncea.

FIG. 22 shows the fatty acid compositions of the seed lipids from fivetransgenic lines expressing Fad6; SA=stearic acid; OA=oleic acid;LA=linoleic acid; GLA=γ-linolenic acid; ALA=α-linolenic acid;SDA=stearidonic acid.

FIG. 23 is a table showing the fatty acid profile of Thraustochytriumsp.

FIG. 24 is a table showing the fatty acid profile of Pythium irregulare.

FIG. 25 is a table showing the conversion of exogenous fatty acids inyeast AMY-2α/pFad5-2.

FIG. 26 is a table showing the accumulation of Δ5-unsaturatedpolymethylene-interrupted fatty acids (Δ5-UPIFAs) in transgenicflaxseeds expressing Fad5-2 under the control of napin (Napin) and flaxseed-specific (Cln) promoters. The fatty acid levels are shown as theweight percentage of the total fatty acids.

FIG. 27 is a table showing the accumulation of Δ6 desaturated fattyacids in transgenic flaxseeds (Solin and Normandy) expressing Fad6 underthe control of the napin promoter. The fatty acid levels are shown asthe weight percentage of the total fatty acids.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the discovery ofnovel fatty acid desaturase family members, referred to interchangeablyherein as “desaturases” or “desaturase” nucleic acid and proteinmolecules (e.g., Fad4, Fad5, Fad5-2, and Fad6). These novel moleculesare members of the fatty acid desaturase family and are expressed inLCPUFAs-producing organisms, e.g., Thraustochytrium, Pythium irregulare,Schizichytrium, and Crythecodinium.

As used herein, the term “fatty acids” is art recognized and includes along-chain hydrocarbon based carboxylic acid. Fatty acids are componentsof many lipids including glycerides. The most common naturally occurringfatty acids are monocarboxylic acids which have an even number of carbonatoms (16 or 18) and which may be saturated or unsaturated.“Unsaturated” fatty acids contain cis double bonds between the carbonatoms. Unsaturated fatty acids encompassed by the present inventioninclude, for example, DHA, GLA, and SDA. “Polyunsaturated” fatty acidscontain more than one double bond and the double bonds are arranged in amethylene interrupted system (—CH═CH—CH₂—CH═CH—).

Fatty acids are described herein by a numbering system in which thenumber before the colon indicates the number of carbon atoms in thefatty acid, whereas the number after the colon is the number of doublebonds that are present. In the case of unsaturated fatty acids, this isfollowed by a number in parentheses that indicates the position of thedouble bonds. Each number in parenthesis is the lower numbered carbonatom of the two connected by the double bond. For example, oleic acidcan be described as 18:1(9) and linoleic acid can be described as18:2(9, 12) indicating 18 carbons, one double bond at carbon 9, twodouble bonds at carbons 9 and 12, respectively.

The controlling steps in the production of unsaturated fatty acids,i.e., the unsaturated fatty acid biosynthetic pathway, are catalyzed bymembrane-associated fatty acid desaturases, e.g., Fad4, Fad5, Fad5-2,and/or Fad6. Specifically, such enzymes catalyze the formation of doublebonds between the carbon atoms of a fatty acid molecule. As used herein,the term “unsaturated fatty acid biosynthetic pathway” refers to aseries of chemical reactions leading to the synthesis of an unsaturatedfatty acid either in vivo or in vitro. Such a pathway includes a seriesof desaturation and elongation steps which generate unsaturated fattyacids and ultimately, long chain polyunsaturated fatty acids. Suchunsaturated fatty acids can include, GLA 18:3 (6,9,12), SDA 18:4(6,9,12,15), AA 20:4 (5,8,11,14), EPA 20:5 (5,8,11,14,17), and DPA 22:5(4,7,10,13,16), and DHA 22:6 (4,7,10,13,16,19).

Desaturases can contain a heme-binding motif and/or about threeconservative histidine motifs, although additional domains may bepresent. Members of the fatty acid desaturase family convert saturatedfatty acids to unsaturated fatty acids, e.g., long chain polyunsaturatedfatty acids (LCPUFAs), which are components of cell membranes of varioustissues and organelles in mammals (nerve, retina, brain and immunecells). Examples of LCPUFA include, among others, docosahexaenoic acid(DHA, 22:6(4,7,10,13,16,19)). Clinical studies have shown that DHA isessential for the growth and development of the brain in infants, andfor maintenance of normal brain function in adults (Martinetz, M. (1992)J. Pediatr. 120:S129-S138). DHA also has effects on photoreceptorfunction involved in the signal transduction process, rhodopsinactivation, and rod and cone development (Giusto, N. M., et al. (2000)Prog. Lipid Res. 39:315-391). In addition, positive effects of DHA werealso found in the treatment of diseases such as hypertension, arthritis,atherosclerosis, depression, thrombosis and cancers (Horrocks, L. A. andYeo, Y. K. (1999) Pharmacol. Res. 40:211-215). Thus, the desaturasemolecules can be used to produce the LCPUFAs useful in treatingdisorders characterized by aberrantly regulated growth, proliferation,or differentiation. Such disorders include cancer, e.g., carcinoma,sarcoma, or leukemia; tumor angiogenesis and metastasis; skeletaldysplasia; hepatic disorders; myelodysplastic syndromes; andhematopoietic and/or myeloproliferative disorders. Other disordersrelated to angiogenesis and which are, therefore, desaturase associateddisorders include hereditary hemorrhagic telangiectasia type 1,fibrodysplasia ossificans progressiva, idiopathic pulmonary fibrosis,and Klippel-Trenaunay-Weber syndrome.

The term “family” when referring to the protein and nucleic acidmolecules of the present invention is intended to mean two or moreproteins or nucleic acid molecules having a common structural domain ormotif and having sufficient amino acid or nucleotide sequence homologyas defined herein. Such family members can be naturally or non-naturallyoccurring and can be from either the same or different species. Forexample, a family can contain a first protein of human origin as well asother distinct proteins of human origin or alternatively, can containhomologues of non-human origin, e.g., rat or mouse proteins. Members ofa family can also have common functional characteristics.

For example, the family of desaturase proteins of the present inventioncomprises one cytochrome b5 heme-binding motif. As used herein, the term“heme-binding motif” is an N-terminal extension of the cytochromeb5-like domain found in front-end desaturases.

In another embodiment, members of the desaturase family of proteinsinclude a “histidine motifs” in the protein, preferably, about three orfour histidine motifs. As used herein, the term “histidine motif”includes a protein domain having at least about two histidine amino acidresidues, preferably about three or four histidine amino acid residues,and is typically found in all microsomal desaturases as the thirdconservative histidine motif.

Examples of cytochrome b5 heme-binding motifs and histidine motifsinclude amino acid residues 41-44, 182-186, 216-223, and 453-462 of SEQID NO:2, amino acid residues 40-43, 171-175, 207-213, and 375-384 of SEQID NO:4, amino acid residues 40-45, 171-176, 208-213, and 395-400 of SEQID NO:6, and amino acid residues 42-47, 178-183, 215-220, and 400-405 ofSEQ ID NO:8, as shown in FIGS. 3 and 6.

Isolated desaturase proteins of the present invention have an amino acidsequence sufficiently homologous to the amino acid sequence of SEQ IDNO:2, 4, 6, or 8 or are encoded by a nucleotide sequence sufficientlyhomologous to SEQ ID NO:1, 3, 5 or 7. As used herein, the term“sufficiently homologous” refers to a first amino acid or nucleotidesequence which contains a sufficient or minimum number of identical orequivalent (e.g., an amino acid residue which has a similar side chain)amino acid residues or nucleotides to a second amino acid or nucleotidesequence such that the first and second amino acid or nucleotidesequences share common structural domains or motifs and/or a commonfunctional activity. For example, amino acid or nucleotide sequenceswhich share common structural domains having at least 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or morehomology or identity across the amino acid sequences of the domains andcontain at least one and preferably two structural domains or motifs,are defined herein as sufficiently homologous. Furthermore, amino acidor nucleotide sequences which share at least 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology oridentity and share a common functional activity are defined herein assufficiently homologous.

In a preferred embodiment, a desaturase protein includes at least one ormore of the following domains or motifs: a heme-binding motif and/or ahistidine motif and has an amino acid sequence at least about 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or morehomologous or identical to the amino acid sequence of SEQ ID NO:2, 4, 6,or 8. In yet another preferred embodiment, a desaturase protein includesat least one or more of the following domains: a heme-binding motifand/or a histidine motif, and is encoded by a nucleic acid moleculehaving a nucleotide sequence which hybridizes under stringenthybridization conditions to a complement of a nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO:1, 3, 5, or 7. Inanother preferred embodiment, a desaturase protein includes at least oneheme-binding motif and/or at least about three histidine motifs, and hasa desaturase activity.

As used interchangeably herein, a “desaturase activity,” “biologicalactivity of a desaturase,” or “functional activity of a desaturase,”includes an activity exerted or mediated by a desaturase protein,polypeptide or nucleic acid molecule on a desaturase responsive cell oron a desaturase substrate, as determined in vivo or in vitro, accordingto standard techniques. In one embodiment, a desaturase activity is adirect activity such as an association with a desaturase targetmolecule. As used herein, a “target molecule” or “binding partner” is amolecule e.g., a molecule involved in the synthesis of unsaturated fattyacids, e.g., an intermediate fatty acid, with which a desaturase proteinbinds or interacts in nature such that a desaturase-mediated function isachieved. A desaturase direct activity also includes the formation of adouble bond between the carbon atoms of a fatty acid molecule to form anunsaturated fatty acid molecule.

The nucleotide sequence of the isolated Thraustochytrium sp. Δ4desaturase, Fad4, cDNA and the predicted amino acid sequence encoded bythe Fad4 cDNA are shown in FIGS. 1A and 1B and in SEQ ID NOs:1 and 2,respectively. The Thraustochytrium sp. Fad4 gene (the open readingframe), which is approximately 1560 nucleotides in length, encodes aprotein having a molecular weight of approximately 59.1 kD and which isapproximately 519 amino acid residues in length.

The nucleotide sequence of the Thraustochytrium sp. Δ5 desaturase, Fad5,cDNA and the predicted amino acid sequence encoded by the Fad5 cDNA areshown in FIGS. 2A and 2B and in SEQ ID NOs:3 and 4, respectively. TheThraustochytrium sp. Fad5 gene, which is approximately 1320 nucleotidesin length, encodes a protein having a molecular weight of approximately49.8 kD and which is approximately 439 amino acid residues in length.

The nucleotide sequence of the Pythium irregulare Δ5 desaturase, Fad5-2,cDNA and the predicted amino acid sequence encoded by the Fad5-2 cDNAare shown in FIGS. 4A and 4B and in SEQ ID NOs:5 and 6, respectively.The Pythium irregulare Fad5-2 gene, which is approximately 1371nucleotides in length, encodes a protein having approximately 456 aminoacid residues in length.

The nucleotide sequence of the Pythium irregulare Δ6 desaturase, Fad6,cDNA and the predicted amino acid sequence encoded by the Fad6 cDNA areshown in FIGS. 5A and 5B and in SEQ ID NOs:7 and 8, respectively. ThePythium irregulare Fad6 gene, which is approximately 1383 nucleotides inlength, encodes a protein having approximately 460 amino acid residuesin length.

Various aspects of the invention are described in further detail in thefollowing subsections:

I. Isolated Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode desaturase proteins or biologically active portions thereof,as well as nucleic acid fragments sufficient for use as hybridizationprobes to identify desaturase-encoding nucleic acid molecules (e.g.,desaturase mRNA) and fragments for use as PCR primers for theamplification or mutation of desaturase nucleic acid molecules. As usedherein, the term “nucleic acid molecule” is intended to include DNAmolecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) andanalogs of the DNA or RNA generated using nucleotide analogs. Thenucleic acid molecule can be single-stranded or double-stranded, butpreferably is double-stranded DNA.

The term “isolated nucleic acid molecule” includes nucleic acidmolecules which are separated from other nucleic acid molecules whichare present in the natural source of the nucleic acid. For example, withregards to genomic DNA, the term “isolated” includes nucleic acidmolecules which are separated from the chromosome with which the genomicDNA is naturally associated. Preferably, an “isolated” nucleic acid isfree of sequences which naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived. Forexample, in various embodiments, the isolated desaturase nucleic acidmolecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5kb or 0.1 kb of nucleotide sequences which naturally flank the nucleicacid molecule in genomic DNA of the cell from which the nucleic acid isderived. Moreover, an “isolated” nucleic acid molecule, such as a cDNAmolecule, can be substantially free of other cellular material, orculture medium when produced by recombinant techniques, or substantiallyfree of chemical precursors or other chemicals when chemicallysynthesized.

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule having the nucleotide sequence of SEQ ID NO:1, 3, 5, 7, or aportion thereof, can be isolated using standard molecular biologytechniques and the sequence information provided herein. Using all or aportion of the nucleic acid sequence of SEQ ID NO:1, 3, 5, or 7, ashybridization probes, desaturase nucleic acid molecules can be isolatedusing standard hybridization and cloning techniques (e.g., as describedin Sambrook, J. et al. Molecular Cloning: A Laboratory Manual. 2^(nd) ,ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1989).

Moreover, a nucleic acid molecule encompassing all or a portion of SEQID NO:1, 3, 5, or 7, can be isolated by the polymerase chain reaction(PCR) using synthetic oligonucleotide primers designed based upon thesequence of SEQ ID NO: 1, 3, 5, or 7.

A nucleic acid of the invention can be amplified using cDNA, mRNA oralternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to desaturase nucleotidesequences can be prepared by standard synthetic techniques, e.g., usingan automated DNA synthesizer.

In another embodiment, the nucleic acid molecule consists of thenucleotide sequence set forth as SEQ ID NO: 1, 3, 5, or 7.

In still another embodiment, an isolated nucleic acid molecule of theinvention comprises a nucleic acid molecule which is a complement of thenucleotide sequence shown in SEQ ID NO: 1, 3, 5, or 7, or a portion ofany of these nucleotide sequences. A nucleic acid molecule which iscomplementary to the nucleotide sequence shown in SEQ ID NO:1, 3, 5, or7 is one which is sufficiently complementary to the nucleotide sequenceshown in SEQ ID NO:1, 3, 5, or 7, such that it can hybridize to thenucleotide sequence shown in SEQ ID NO: 1, 3, 5, or 7, thereby forming astable duplex.

In still another embodiment, an isolated nucleic acid molecule of thepresent invention comprises a nucleotide sequence which is at leastabout 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99% or more identical to the nucleotide sequence shown in SEQ ID NO: 1,3, 5, or 7 (e.g., to the entire length of the nucleotide sequence), or aportion or complement of any of these nucleotide sequences. In oneembodiment, a nucleic acid molecule of the present invention comprises anucleotide sequence which is at least (or no greater than) 50-100,100-250, 250-500, 500-750, 750-1000, 1000-1250, 1250-1500, 1500-1750,1750-2000, 2000-2250, 2250-2500, 2500-2750, 2750-3000, 3250-3500,3500-3750 or more nucleotides in length and hybridizes under stringenthybridization conditions to a complement of a nucleic acid molecule ofSEQ ID NO:1, 3, 5, or 7.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the nucleic acid sequence of SEQ ID NO:1, 3, 5, or 7, forexample, a fragment which can be used as a probe or primer or a fragmentencoding a portion of a desaturase protein, e.g., a biologically activeportion of a desaturase protein. The nucleotide sequence determined fromthe cloning of the desaturase gene allows for the generation of probesand primers designed for use in identifying and/or cloning otherdesaturase family members, as well as desaturase homologues from otherspecies. The probe/primer (e.g., oligonucleotide) typically comprisessubstantially purified oligonucleotide. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12 or 15, preferably about 20 or25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75consecutive nucleotides of a sense sequence of SEQ ID NO: 1, 3, 5 or 7,of an anti-sense sequence of SEQ ID NO:1, 3, 5, or 7, or of a naturallyoccurring allelic variant or mutant of SEQ ID NO: 1, 3, 5, or 7.

Exemplary probes or primers are at least (or no greater than) 12 or 15,20 or 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or more nucleotides inlength and/or comprise consecutive nucleotides of an isolated nucleicacid molecule described herein. Also included within the scope of thepresent invention are probes or primers comprising contiguous orconsecutive nucleotides of an isolated nucleic acid molecule describedherein, but for the difference of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 baseswithin the probe or primer sequence. Probes based on the desaturasenucleotide sequences can be used to detect (e.g., specifically detect)transcripts or genomic sequences encoding the same or homologousproteins. In preferred embodiments, the probe further comprises a labelgroup attached thereto, e.g., the label group can be a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. In anotherembodiment a set of primers is provided, e.g., primers suitable for usein a PCR, which can be used to amplify a selected region of a desaturasesequence, e.g., a domain, region, site or other sequence describedherein. The primers should be at least 5, 10, or 50 base pairs in lengthand less than 100, or less than 200, base pairs in length. The primersshould be identical, or differ by no greater than 1, 2, 3, 4, 5, 6, 7,8, 9 or 10 bases when compared to a sequence disclosed herein or to thesequence of a naturally occurring variant. Such probes can be used as apart of a diagnostic test kit for identifying cells or tissue whichmisexpress a desaturase protein, such as by measuring a level of adesaturase-encoding nucleic acid in a sample of cells from a subject,e.g., detecting desaturase mRNA levels or determining whether a genomicdesaturase gene has been mutated or deleted.

A nucleic acid fragment encoding a “biologically active portion of adesaturase protein” can be prepared by isolating a portion of thenucleotide sequence of SEQ ID NO: 1, 3, 5, or 7, which encodes apolypeptide having a desaturase biological activity (the biologicalactivities of the desaturase proteins are described herein), expressingthe encoded portion of the desaturase protein (e.g., by recombinantexpression in vitro) and assessing the activity of the encoded portionof the desaturase protein. In an exemplary embodiment, the nucleic acidmolecule is at least 50-100, 100-250, 250-500, 500-700, 750-1000,1000-1250, 1250-1500, 1500-1750, 1750-2000, 2000-2250, 2250-2500,2500-2750, 2750-3000, 3250-3500, 3500-3750 or more nucleotides in lengthand encodes a protein having a desaturase activity (as describedherein).

The invention further encompasses nucleic acid molecules that differfrom the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, or 7 due todegeneracy of the genetic code and thus encode the same desaturaseproteins as those encoded by the nucleotide sequence shown in SEQ ID NO:1, 3, 5, or 7. In another embodiment, an isolated nucleic acid moleculeof the invention has a nucleotide sequence encoding a protein having anamino acid sequence which differs by at least 1, but no greater than 5,10, 20, 50 or 100 amino acid residues from the amino acid sequence shownin SEQ ID NO:2, 4, 6, or 8. In yet another embodiment, the nucleic acidmolecule encodes the amino acid sequence of human desaturase. If analignment is needed for this comparison, the sequences should be alignedfor maximum homology.

Nucleic acid variants can be naturally occurring, such as allelicvariants (same locus), homologues (different locus), and orthologues(different organism) or can be non naturally occurring. Non-naturallyoccurring variants can be made by mutagenesis techniques, includingthose applied to polynucleotides, cells, or organisms. The variants cancontain nucleotide substitutions, deletions, inversions and insertions.Variation can occur in either or both the coding and non-coding regions.The variations can produce both conservative and non-conservative aminoacid substitutions (as compared in the encoded product).

Allelic variants result, for example, from DNA sequence polymorphismswithin a population (e.g., the human population) that lead to changes inthe amino acid sequences of the desaturase proteins. Such geneticpolymorphism in the desaturase genes may exist among individuals withina population due to natural allelic variation. As used herein, the terms“gene” and “recombinant gene” refer to nucleic acid molecules whichinclude an open reading frame encoding a desaturase protein, e.g.,oilseed desaturase protein, and can further include non-codingregulatory sequences, and introns.

Accordingly, in one embodiment, the invention features isolated nucleicacid molecules which encode a naturally occurring allelic variant of apolypeptide comprising the amino acid sequence of SEQ ID NO:2, 4, 6, or8, wherein the nucleic acid molecule hybridizes to a complement of anucleic acid molecule comprising SEQ ID NO: 1, 3, 5, or 7, for example,under stringent hybridization conditions.

Allelic variants of desaturase, e.g., Fad4, Fad5, Fad5-2, or Fad6,include both functional and non-functional desaturase proteins.Functional allelic variants are naturally occurring amino acid sequencevariants of the desaturase protein that maintain the ability to, e.g.,(i) interact with a desaturase substrate or target molecule (e.g., afatty acid, e.g., 22:5(n−3)); and/or (ii) form a double bond betweencarbon atoms in a desaturase substrate or target molecule. The fattyacids produced by the nucleic acid and protein molecules of the presentinvention are also useful in treating disorders such as aging, stress,diabetes, cancer, inflammatory disorders (e.g., arthritis, eczema), andcardiovascular disorders. Functional allelic variants will typicallycontain only a conservative substitution of one or more amino acids ofSEQ ID NO:2, 4, 6, or 8, or a substitution, deletion or insertion ofnon-critical residues in non-critical regions of the protein.

Non-functional allelic variants are naturally occurring amino acidsequence variants of the desaturase protein, e.g., Fad4, Fad5, Fad5-2,or Fad6, that do not have the ability to, e.g., (i) interact with adesaturase substrate or target molecule (e.g., an intermediate fattyacid, such as 18:4(6,9,12,15)); and/or (ii) form a double bond betweencarbon atoms in a desaturase substrate or target molecule.Non-functional allelic variants will typically contain anon-conservative substitution, a deletion, or insertion, or prematuretruncation of the amino acid sequence of SEQ ID NO:2, 4, 6, or 8, or asubstitution, insertion, or deletion in critical residues or criticalregions of the protein.

The present invention further provides orthologues (e.g., humanorthologues of the desaturase proteins). Orthologues of theThraustochytrium sp. and Pythium irregulare desaturase proteins areproteins that are isolated from other organisms and possess the samedesaturase substrate or target molecule binding mechanisms, double bondforming mechanisms, modulating mechanisms of growth and development ofthe brain in infants, maintenance mechanisms of normal brain function inadults, ability to affect photoreceptor function involved in the signaltransduction process, ability to affect rhodopsin activation,development mechanisms of rods and/or cones, and/or modulatingmechanisms of cellular growth and/or proliferation of the non-humandesaturase proteins. Orthologues of the Thraustochytrium sp. and Pythiumirregulare desaturase proteins can readily be identified as comprisingan amino acid sequence that is substantially homologous to SEQ ID NO:2,4, 6, or 8.

Moreover, nucleic acid molecules encoding other desaturase familymembers and, thus, which have a nucleotide sequence which differs fromthe desaturase sequences of SEQ ID NO: 1, 3, 5, or 7 are intended to bewithin the scope of the invention. For example, another desaturase cDNAcan be identified based on the nucleotide sequence of Fad4, Fad5,Fad5-2, or Fad6. Moreover, nucleic acid molecules encoding desaturaseproteins from different species, and which, thus, have a nucleotidesequence which differs from the desaturase sequences of SEQ ID NO: 1, 3,5, or 7 are intended to be within the scope of the invention. Forexample, Schizochytrium or Crythecodinium desaturase cDNA can beidentified based on the nucleotide sequence of a Fad4, Fad5, Fad5-2, orFad6.

Nucleic acid molecules corresponding to natural allelic variants andhomologues of the desaturase cDNAs of the invention can be isolatedbased on their homology to the desaturase nucleic acids disclosed hereinusing the cDNAs disclosed herein, or a portion thereof, as ahybridization probe according to standard hybridization techniques understringent hybridization conditions.

Orthologues, homologues and allelic variants can be identified usingmethods known in the art (e.g., by hybridization to an isolated nucleicacid molecule of the present invention, for example, under stringenthybridization conditions). In one embodiment, an isolated nucleic acidmolecule of the invention is at least 15, 20, 25, 30 or more nucleotidesin length and hybridizes under stringent conditions to the nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO:1, 3, 5, or 7.In other embodiment, the nucleic acid is at least 50-100, 100-250,250-500, 500-700, 750-1000, 1000-1250, 1250-1500, 1500-1750, 1750-2000,2000-2250, 2250-2500, 2500-2750, 2750-3000, 3250-3500, 3500-3750 or morenucleotides in length.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences that are significantly identical orhomologous to each other remain hybridized to each other. Preferably,the conditions are such that sequences at least about 70%, morepreferably at least about 80%, even more preferably at least about 85%or 90% identical to each other remain hybridized to each other. Suchstringent conditions are known to those skilled in the art and can befound in Current Protocols in Molecular Biology, Ausubel et al., eds.,John Wiley & Sons, Inc. (1995), sections 2, 4, and 6. Additionalstringent conditions can be found in Molecular Cloning: A LaboratoryManual, Sambrook et al., Cold Spring Harbor Press, Cold Spring Harbor,N.Y. (1989), chapters 7, 9, and 11. A preferred, non-limiting example ofstringent hybridization conditions includes hybridization in 4× sodiumchloride/sodium citrate (SSC), at about 65-70° C. (or alternativelyhybridization in 4×SSC plus 50% formamide at about 42-50° C.) followedby one or more washes in 1×SSC, at about 65-70° C. A preferred,non-limiting example of highly stringent hybridization conditionsincludes hybridization in 1×SSC, at about 65-70° C. (or alternativelyhybridization in 1×SSC plus 50% formamide at about 42-50° C.) followedby one or more washes in 0.3×SSC, at about 65-70° C. A preferred,non-limiting example of reduced stringency hybridization conditionsincludes hybridization in 4×SSC, at about 50-60° C. (or alternativelyhybridization in 6×SSC plus 50% formamide at about 40-45° C.) followedby one or more washes in 2×SSC, at about 50-60° C. Ranges intermediateto the above-recited values, e.g., at 65-70° C. or at 42-50° C. are alsointended to be encompassed by the present invention. SSPE (1×SSPE is0.15M NaCl, 10 mM NaH₂PO₄, and 1.25 mM EDTA, pH 7.4) can be substitutedfor SSC (1×SSC is 0.15M NaCl and 15 mM sodium citrate) in thehybridization and wash buffers; washes are performed for 15 minutes eachafter hybridization is complete. The hybridization temperature forhybrids anticipated to be less than 50 base pairs in length should be5-10° C. less than the melting temperature (T_(m)) of the hybrid, whereT_(m) is determined according to the following equations. For hybridsless than 18 base pairs in length, T_(m)(° C.)=2(# of A+T bases)+4(# ofG+C bases). For hybrids between 18 and 49 base pairs in length, T_(m)(°C.)=81.5+16.6(log₁₀[Na⁺])+0.41(% G+C)−(600/N), where N is the number ofbases in the hybrid, and [Na⁺] is the concentration of sodium ions inthe hybridization buffer ([Na⁺] for 1×SSC=0.165 M). It will also berecognized by the skilled practitioner that additional reagents may beadded to hybridization and/or wash buffers to decrease non-specifichybridization of nucleic acid molecules to membranes, for example,nitrocellulose or nylon membranes, including but not limited to blockingagents (e.g., BSA or salmon or herring sperm carrier DNA), detergents(e.g., SDS), chelating agents (e.g., EDTA), Ficoll, PVP and the like.When using nylon membranes, in particular, an additional preferred,non-limiting example of stringent hybridization conditions ishybridization in 0.25-0.5M NaH₂PO₄, 7% SDS at about 65° C., followed byone or more washes at 0.02M NaH₂PO₄, 1% SDS at 65° C. (see e.g., Churchand Gilbert (1984) Proc. Natl. Acad. Sci. USA 81:1991-1995), oralternatively 0.2×SSC, 1% SDS.

Preferably, an isolated nucleic acid molecule of the invention thathybridizes under stringent conditions to the sequence of SEQ ID NO:1, 3,5, or 7 corresponds to a naturally-occurring nucleic acid molecule. Asused herein, a “naturally-occurring” nucleic acid molecule refers to anRNA or DNA molecule having a nucleotide sequence that occurs in nature(e.g., encodes a natural protein).

In addition to naturally-occurring allelic variants of the desaturasesequences that may exist in the population, the skilled artisan willfurther appreciate that changes can be introduced by mutation into thenucleotide sequences of SEQ ID NO: 1, 3, 5, or 7, thereby leading tochanges in the amino acid sequence of the encoded desaturase proteins,without altering the functional ability of the desaturase proteins. Forexample, nucleotide substitutions leading to amino acid substitutions at“non-essential” amino acid residues can be made in the sequence of SEQID NO: 1, 3, 5, or 7. A “non-essential” amino acid residue is a residuethat can be altered from the wild-type sequence of Fad4, Fad5, Fad5-2,or Fad6 (e.g., the sequence of SEQ ID NO:2, 4, 6, or 8) without alteringthe biological activity, whereas an “essential” amino acid residue isrequired for biological activity. For example, amino acid residues thatare conserved among the desaturase proteins of the present invention,e.g., those present in a heme-binding motif or a histidine motif, arepredicted to be particularly unamenable to alteration. Furthermore,additional amino acid residues that are conserved between the desaturaseproteins of the present invention and other members of the fatty aciddesaturase family are not likely to be amenable to alteration.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding desaturase proteins that contain changes in aminoacid residues that are not essential for activity. Such desaturaseproteins differ in amino acid sequence from SEQ ID NO:2, 4, 6, or 8, yetretain biological activity. In one embodiment, the isolated nucleic acidmolecule comprises a nucleotide sequence encoding a protein, wherein theprotein comprises an amino acid sequence at least about 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% 99% or more homologousto SEQ ID NO: 2, 4, 6, or 8, e.g., to the entire length of SEQ ID NO:2,5, 8, or 11.

An isolated nucleic acid molecule encoding a desaturase proteinhomologous to the protein of SEQ ID NO: 2, 4, 6, or 8 can be created byintroducing one or more nucleotide substitutions, additions or deletionsinto the nucleotide sequence of SEQ ID NO:1, 3, 5, or 7, such that oneor more amino acid substitutions, additions or deletions are introducedinto the encoded protein. Mutations can be introduced into SEQ ID NO:1,3, 5, or 7 by standard techniques, such as site-directed mutagenesis andPCR-mediated mutagenesis. Preferably, conservative amino acidsubstitutions are made at one or more predicted non-essential amino acidresidues. A “conservative amino acid substitution” is one in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a desaturase protein ispreferably replaced with another amino acid residue from the same sidechain family. Alternatively, in another embodiment, mutations can beintroduced randomly along all or part of a desaturase coding sequence,such as by saturation mutagenesis, and the resultant mutants can bescreened for desaturase biological activity to identify mutants thatretain activity. Following mutagenesis of SEQ ID NO:1, 3, 5, or 7, theencoded protein can be expressed recombinantly and the activity of theprotein can be determined.

In a preferred embodiment, a mutant desaturase protein can be assayedfor the ability to (i) interact with a desaturase substrate or targetmolecule (e.g., an intermediate fatty acid); and/or (ii) form a doublebond between carbon atoms in a desaturase substrate or target molecule.

II. Isolated Desaturase Proteins

One aspect of the invention pertains to isolated or recombinantdesaturase proteins and polypeptides, and biologically active portionsthereof. In one embodiment, native desaturase proteins can be isolatedfrom cells or tissue sources by an appropriate purification scheme usingstandard protein purification techniques. In another embodiment,desaturase proteins are produced by recombinant DNA techniques.Alternative to recombinant expression, a desaturase protein orpolypeptide can be synthesized chemically using standard peptidesynthesis techniques.

An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which thedesaturase protein is derived, or substantially free from chemicalprecursors or other chemicals when chemically synthesized. The language“substantially free of cellular material” includes preparations ofdesaturase protein in which the protein is separated from cellularcomponents of the cells from which it is isolated or recombinantlyproduced. In one embodiment, the language “substantially free ofcellular material” includes preparations of desaturase protein havingless than about 80%, 70%, 60%, 50%, 40%, or 30% (by dry weight) ofnon-desaturase protein (also referred to herein as a “contaminatingprotein”), more preferably less than about 20% of non-desaturaseprotein, still more preferably less than about 10% of non-desaturaseprotein, and most preferably less than about 5% non-desaturase protein.When the desaturase protein or biologically active portion thereof isrecombinantly produced, it is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,more preferably less than about 10%, and most preferably less than about5% of the volume of the protein preparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of desaturase protein in which theprotein is separated from chemical precursors or other chemicals whichare involved in the synthesis of the protein. In one embodiment, thelanguage “substantially free of chemical precursors or other chemicals”includes preparations of desaturase protein having less than about 30%(by dry weight) of chemical precursors or non-desaturase chemicals, morepreferably less than about 20% chemical precursors or non-desaturasechemicals, still more preferably less than about 10% chemical precursorsor non-desaturase chemicals, and most preferably less than about 5%chemical precursors or non-desaturase chemicals. It should be understoodthat the proteins or this invention can also be in a form which isdifferent than their corresponding naturally occurring proteins and/orwhich is still in association with at least some cellular components.For example, the protein can be associated with a cellular membrane.

As used herein, a “biologically active portion” of a desaturase proteinincludes a fragment of a desaturase protein which participates in aninteraction between a desaturase molecule and a non-desaturase molecule(e.g., a desaturase substrate such as fatty acid). Biologically activeportions of a desaturase protein include peptides comprising amino acidsequences sufficiently homologous to or derived from the desaturaseamino acid sequences, e.g., the amino acid sequences shown in SEQ IDNO:2, 4, 6, or 8 which include sufficient amino acid residues to exhibitat least one activity of a desaturase protein. Typically, biologicallyactive portions comprise a domain or motif with at least one activity ofthe desaturase protein; the ability to (i) interact with a desaturasesubstrate or target molecule (e.g., an intermediate fatty acid); and/or(ii) form a double bond between carbon atoms in a desaturase substrateor target molecule. A biologically active portion of a desaturaseprotein can be a polypeptide which is, for example, 10, 25, 50, 75, 100,125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200 or more aminoacids in length.

In one embodiment, a biologically active portion of a desaturase proteincomprises a heme-binding motif and/or at least one histidine motifs,preferably about three histidine motifs. Moreover, other biologicallyactive portions, in which other regions of the protein are deleted, canbe prepared by recombinant techniques and evaluated for one or more ofthe functional activities of a native desaturase protein.

In a preferred embodiment, a desaturase protein has an amino acidsequence shown in SEQ ID NO: 2, 4, 6, or 8. In other embodiments, thedesaturase protein is substantially identical to SEQ ID NO: 2, 4, 6, or8 and retains the functional activity of the protein of SEQ ID NO: 2, 4,6, or 8, yet differs in amino acid sequence due to natural allelicvariation or mutagenesis, as described in detail in subsection I above.In another embodiment, the desaturase protein is a protein whichcomprises an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ IDNO: 2, 4, 6, or 8.

In another embodiment, the invention features a desaturase protein whichis encoded by a nucleic acid molecule consisting of a nucleotidesequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or more identical to a nucleotide sequence ofSEQ ID NO:1, 3, 5, or 7, or a complement thereof. This invention furtherfeatures a desaturase protein which is encoded by a nucleic acidmolecule consisting of a nucleotide sequence which hybridizes understringent hybridization conditions to a complement of a nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO:1, 3, 5, or 7,or a complement thereof.

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, or 90% of the length of the referencesequence (e.g., when aligning a second sequence to the Fad4 amino acidsequence of SEQ ID NO:2 having 519 amino acid residues, at least 156,preferably at least 208, more preferably at least 260, even morepreferably at least 311, and even more preferably at least 363, 415, or467 amino acid residues are aligned; when aligning a second sequence tothe Fad5 amino acid sequence of SEQ ID NO:4 having 439 amino acidresidues, at least 132, preferably at least 176, more preferably atleast 220, even more preferably at least 263, and even more preferablyat least 307, 351, or 395 amino acid residues are aligned; when aligninga second sequence to the Fad5-2 amino acid sequence of SEQ ID NO:6having 456 amino acid residues, at least 137, preferably at least 182,more preferably at least 228, even more preferably at least 273, andeven more preferably at least 319, 365, or 419 amino acid residues arealigned; when aligning a second sequence to the Fad6 amino acid sequenceof SEQ ID NO:8 having 460 amino acid residues, at least 138, preferablyat least 184, more preferably at least 230, even more preferably atleast 276, and even more preferably at least 322, 368, or 414 amino acidresidues are aligned). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein amino acid or nucleic acid “identity” is equivalent to aminoacid or nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch (J.Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporatedinto the GAP program in the GCG software package, using either a Blossum62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6,or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet anotherpreferred embodiment, the percent identity between two nucleotidesequences is determined using the GAP program in the GCG softwarepackage, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60,70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A preferred,non-limiting example of parameters to be used in conjunction with theGAP program include a Blosum 62 scoring matrix with a gap penalty of 12,a gap extend penalty of 4, and a frameshift gap penalty of 5.

In another embodiment, the percent identity between two amino acid ornucleotide sequences is determined using the algorithm of Meyers andMiller (Comput. Appl. Biosci., 4:11-17 (1988)) which has beenincorporated into the ALIGN program (version 2.0 or version 2.0U), usinga PAM120 weight residue table, a gap length penalty of 12 and a gappenalty of 4.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify other family members or relatedsequences. Such searches can be performed using the NBLAST and XBLASTprograms (version 2.0) of Altschul et al. (1990) J. Mol. Biol.215:403-10. BLAST nucleotide searches can be performed with the NBLASTprogram, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to desaturase nucleic acid molecules of the invention. BLASTprotein searches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to desaturaseprotein molecules of the invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST can be utilized as described inAltschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. Whenutilizing BLAST and Gapped BLAST programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used.

III. Methods of Producing Unsaturated Fatty Acids

The present invention provides new and improved methods of producingunsaturated fatty acids, e.g., LCPUFAs, such as, DHA (docosahexaenoicacid, 22:6 (n−6)), DPA (docosapentaenoic acid, 22:5 (n−6)), AA(Arachidonic acid, 20:4 (n−6)) and EPA (eicosapentaenioc acid,20:5(n−3)).

A. Recombinant Cells and Methods for Culturing Cells

The present invention further features recombinant vectors that includenucleic acid sequences that encode the gene products as describedherein, preferably Fad4, Fad5, Fad5-2, and Fad6 gene products. The termrecombinant vector includes a vector (e.g., plasmid) that has beenaltered, modified or engineered such that it contains greater, fewer ordifferent nucleic acid sequences than those included in the nativevector or plasmid. In one embodiment, a recombinant vector includes thenucleic acid sequence encoding at least one fatty acid desaturase enzymeoperably linked to regulatory sequences. The phrase “operably linked toregulatory sequence(s)” means that the nucleotide sequence of interestis linked to the regulatory sequence(s) in a manner which allows forexpression (e.g., enhanced, increased, constitutive, basal, attenuated,decreased or repressed expression) of the nucleotide sequence,preferably expression of a gene product encoded by the nucleotidesequence (e.g., when the recombinant vector is introduced into a cell).Exemplary vectors are described in further detail herein as well as in,for example, Frascotti et al., U.S. Pat. No. 5,721,137, the contents ofwhich are incorporated herein by reference.

The term “regulatory sequence” includes nucleic acid sequences whichaffect (e.g., modulate or regulate) expression of other (non-regulatory)nucleic acid sequences. In one embodiment, a regulatory sequence isincluded in a recombinant vector in a similar or identical positionand/or orientation relative to a particular gene of interest as isobserved for the regulatory sequence and gene of interest as it appearsin nature, e.g., in a native position and/or orientation. For example, agene of interest (e.g., a Fad4, Fad5, Fad5-2, or Fad6 gene) can beincluded in a recombinant vector operably linked to a regulatorysequence which accompanies or is adjacent to the gene in the naturalorganism (e.g., operably linked to “native” Fad4, Fad5, Fad5-2, or Fad6regulatory sequence (e.g., to the “native” Fad4, Fad5, Fad5-2, or Fad6promoter). Alternatively, a gene of interest (e.g., a Fad4, Fad5,Fad5-2, or Fad6 gene) can be included in a recombinant vector operablylinked to a regulatory sequence which accompanies or is adjacent toanother (e.g., a different) gene in the natural organism. For example, aFad4, Fad5, Fad5-2, or Fad6 gene can be included in a vector operablylinked to non-Fad4, Fad5, Fad5-2, or Fad6 regulatory sequences.Alternatively, a gene of interest (e.g., a Fad4, Fad5, Fad5-2, or Fad6gene) can be included in a vector operably linked to a regulatorysequence from another organism. For example, regulatory sequences fromother microbes (e.g., other bacterial regulatory sequences,bacteriophage regulatory sequences and the like) can be operably linkedto a particular gene of interest.

Preferred regulatory sequences include promoters, enhancers, terminationsignals and other expression control elements (e.g., binding sites fortranscriptional and/or translational regulatory proteins, for example,in the transcribed mRNA). Such regulatory sequences are described, forexample, in Sambrook, J., Fritsh, E. F., and Maniatis, T. MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.Regulatory sequences include those which direct constitutive expressionof a nucleotide sequence in a cell (e.g., constitutive promoters andstrong constitutive promoters), those which direct inducible expressionof a nucleotide sequence in a cell (e.g., inducible promoters, forexample, xylose inducible promoters) and those which attenuate orrepress expression of a nucleotide sequence in a cell (e.g., attenuationsignals or repressor sequences). It is also within the scope of thepresent invention to regulate expression of a gene of interest byremoving or deleting regulatory sequences. For example, sequencesinvolved in the negative regulation of transcription can be removed suchthat expression of a gene of interest is enhanced.

In one embodiment, a recombinant vector of the present inventionincludes nucleic acid sequences that encode at least one gene product(e.g., Fad4, Fad5, Fad5-2, or Fad6) operably linked to a promoter orpromoter sequence.

In yet another embodiment, a recombinant vector of the present inventionincludes a terminator sequence or terminator sequences (e.g.,transcription terminator sequences). The term “terminator sequences”includes regulatory sequences which serve to terminate transcription ofmRNA. Terminator sequences (or tandem transcription terminators) canfurther serve to stabilize mRNA (e.g., by adding structure to mRNA), forexample, against nucleases.

In yet another embodiment, a recombinant vector of the present inventionincludes antibiotic resistance sequences. The term “antibioticresistance sequences” includes sequences which promote or conferresistance to antibiotics on the host organism. In one embodiment, theantibiotic resistance sequences are selected from the group consistingof cat (chloramphenicol resistance), tet (tetracycline resistance)sequences, erm (erythromycin resistance) sequences, neo (neomycinresistance) sequences and spec (spectinomycin resistance) sequences.Recombinant vectors of the present invention can further includehomologous recombination sequences (e.g., sequences designed to allowrecombination of the gene of interest into the chromosome of the hostorganism). For example, amyE sequences can be used as homology targetsfor recombination into the host chromosome.

The term “manipulated cell” includes a cell that has been engineered(e.g., genetically engineered) or modified such that the cell has atleast one fatty acid desaturase, e.g., Fad4, Fad5, Fad5-2, and/or Fad6,such that an unsaturated fatty acid is produced. Modification orengineering of such microorganisms can be according to any methodologydescribed herein including, but not limited to, deregulation of abiosynthetic pathway and/or overexpression of at least one biosyntheticenzyme. A “manipulated” enzyme (e.g., a “manipulated” biosyntheticenzyme) includes an enzyme, the expression or production of which hasbeen altered or modified such that at least one upstream or downstreamprecursor, substrate or product of the enzyme is altered or modified,for example, as compared to a corresponding wild-type or naturallyoccurring enzyme.

The term “overexpressed” or “overexpression” includes expression of agene product (e.g., a fatty acid desaturase) at a level greater thanthat expressed prior to manipulation of the cell or in a comparable cellwhich has not been manipulated. In one embodiment, the cell can begenetically manipulated (e.g., genetically engineered) to overexpress alevel of gene product greater than that expressed prior to manipulationof the cell or in a comparable cell which has not been manipulated.Genetic manipulation can include, but is not limited to, altering ormodifying regulatory sequences or sites associated with expression of aparticular gene (e.g., by adding strong promoters, inducible promotersor multiple promoters or by removing regulatory sequences such thatexpression is constitutive), modifying the chromosomal location of aparticular gene, altering nucleic acid sequences adjacent to aparticular gene such as a ribosome binding site or transcriptionterminator, increasing the copy number of a particular gene, modifyingproteins (e.g., regulatory proteins, suppressors, enhancers,transcriptional activators and the like) involved in transcription of aparticular gene and/or translation of a particular gene product, or anyother conventional means of deregulating expression of a particular generoutine in the art (including but not limited to use of antisensenucleic acid molecules, for example, to block expression of repressorproteins).

In another embodiment, the cell can be physically or environmentallymanipulated to overexpress a level of gene product greater than thatexpressed prior to manipulation of the cell or in a comparable cellwhich has not been manipulated. For example, a cell can be treated withor cultured in the presence of an agent known or suspected to increasetranscription of a particular gene and/or translation of a particulargene product such that transcription and/or translation are enhanced orincreased. Alternatively, a cell can be cultured at a temperatureselected to increase transcription of a particular gene and/ortranslation of a particular gene product such that transcription and/ortranslation are enhanced or increased.

The term “deregulated” or “deregulation” includes the alteration ormodification of at least one gene in a cell that encodes an enzyme in abiosynthetic pathway, such that the level or activity of thebiosynthetic enzyme in the cell is altered or modified. Preferably, atleast one gene that encodes an enzyme in a biosynthetic pathway isaltered or modified such that the gene product is enhanced or increased.The phrase “deregulated pathway” can also include a biosynthetic pathwayin which more than one gene that encodes an enzyme in a biosyntheticpathway is altered or modified such that the level or activity of morethan one biosynthetic enzyme is altered or modified. The ability to“deregulate” a pathway (e.g., to simultaneously deregulate more than onegene in a given biosynthetic pathway) in a cell arises from theparticular phenomenon of cells in which more than one enzyme (e.g., twoor three biosynthetic enzymes) are encoded by genes occurring adjacentto one another on a contiguous piece of genetic material termed an“operon”.

The term “operon” includes a coordinated unit of gene expression thatcontains a promoter and possibly a regulatory element associated withone or more, preferably at least two, structural genes (e.g., genesencoding enzymes, for example, biosynthetic enzymes). Expression of thestructural genes can be coordinately regulated, for example, byregulatory proteins binding to the regulatory element or byanti-termination of transcription. The structural genes can betranscribed to give a single mRNA that encodes all of the structuralproteins. Due to the coordinated regulation of genes included in anoperon, alteration or modification of the single promoter and/orregulatory element can result in alteration or modification of each geneproduct encoded by the operon. Alteration or modification of theregulatory element can include, but is not limited to removing theendogenous promoter and/or regulatory element(s), adding strongpromoters, inducible promoters or multiple promoters or removingregulatory sequences such that expression of the gene products ismodified, modifying the chromosomal location of the operon, alteringnucleic acid sequences adjacent to the operon or within the operon suchas a ribosome binding site, increasing the copy number of the operon,modifying proteins (e.g., regulatory proteins, suppressors, enhancers,transcriptional activators and the like) involved in transcription ofthe operon and/or translation of the gene products of the operon, or anyother conventional means of deregulating expression of genes routine inthe art (including but not limited to use of antisense nucleic acidmolecules, for example, to block expression of repressor proteins).Deregulation can also involve altering the coding region of one or moregenes to yield, for example, an enzyme that is feedback resistant or hasa higher or lower specific activity.

A particularly preferred “recombinant” cell of the present invention hasbeen genetically engineered to overexpress a plant-derived gene or geneproduct or an microorganismally-derived gene or gene product. The term“plant-derived,” “microorganismally-derived,” or “derived-from,” forexample, includes a gene which is naturally found in a microorganism ora plant, e.g., an oilseed plant, or a gene product (e.g., Fad4, Fad5,Fad5-2, or Fad6) or which is encoded by a plant gene or a gene from amicroorganism (e.g., encoded SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, orSEQ ID NO:7).

The methodologies of the present invention feature recombinant cellswhich overexpress at least one fatty acid desaturase. In one embodiment,a recombinant cell of the present invention has been geneticallyengineered to overexpress a Thrauschytrium sp. fatty acid desaturase(e.g., has been engineered to overexpress at least one of Thrauschytriumsp. Δ4 or Δ5 desaturase (the Fad4 or Fad5 gene product) (e.g., a fattyacid desaturase having the amino acid sequence of SEQ ID NO:2 or 4 orencoded by the nucleic acid sequence of SEQ ID NO:1 or 3).

In another embodiment, a recombinant cell of the present invention hasbeen genetically engineered to overexpress a Pythium irregulare Δ5 or Δ6desaturase (the Fad5-2 or Fad6 gene product) (e.g., a fatty aciddesaturase having the amino acid sequence of SEQ ID NO:6 or 8 or encodedby a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:5or 7).

In another embodiment, the invention features a cell (e.g., a microbialcell) that has been transformed with a vector comprising a fatty aciddesaturase nucleic acid sequence (e.g., a fatty acid desaturase nucleicacid sequence as set forth in SEQ ID NO:1, 3, 5, or 7).

Another aspect of the present invention features a method of modulatingthe production of fatty acids comprising culturing cells transformed bythe nucleic acid molecules of the present invention (e.g., a desaturase)such that modulation of fatty acid production occurs (e.g., productionof unsaturated fatty acids is enhanced). The method of culturing cellstransformed by the nucleic acid molecules of the present invention(e.g., Fad4, Fad5, Fad5-2, and Fad6) to modulate the production of fattyacids is referred to herein as “biotransformation.” Thebiotransformation processes can utilize recombinant cells and/ordesaturases described herein. The term “biotransformation process,” alsoreferred to herein as “bioconversion processes,” includes biologicalprocesses which result in the production (e.g., transformation orconversion) of any compound (e.g., substrate, intermediate, or product)which is upstream of a fatty acid desaturase to a compound (e.g.,substrate, intermediate, or product) which is downstream of a fatty aciddesaturase, in particular, an unsaturated fatty acid. In one embodiment,the invention features a biotransformation process for the production ofan unsaturated fatty acid comprising contacting a cell whichoverexpresses at least one fatty acid desaturase with at least oneappropriate substrate under conditions such that an unsaturated fattyacid is produced and, optionally, recovering the fatty acid. In apreferred embodiment, the invention features a biotransformation processfor the production of unsaturated fatty acids comprising contacting acell which overexpresses Fad4, Fad5, Fad5-2, or Fad6 with an appropriatesubstrate (e.g., an intermediate fatty acid) under conditions such thatan unsaturated fatty acid (e.g., DHA, SDA, or GLA) is produced and,optionally, recovering the unsaturated fatty acid. Conditions underwhich an unsaturated fatty acid is produced can include any conditionswhich result in the desired production of an unsaturated fatty acid.

The cell(s) and/or enzymes used in the biotransformation reactions arein a form allowing them to perform their intended function (e.g.,producing a desired fatty acids). The cells can be whole cells, or canbe only those portions of the cells necessary to obtain the desired endresult. The cells can be suspended (e.g., in an appropriate solutionsuch as buffered solutions or media), rinsed (e.g., rinsed free of mediafrom culturing the cell), acetone-dried, immobilized (e.g., withpolyacrylamide gel or k-carrageenan or on synthetic supports, forexample, beads, matrices and the like), fixed, cross-linked orpermeablized (e.g., have permeablized membranes and/or walls such thatcompounds, for example, substrates, intermediates or products can moreeasily pass through said membrane or wall). The type of cell can be anycell capable of being used within the methods of the invention, e.g.,plant, animal, or microbial cells.

An important aspect of the present invention involves growing therecombinant plant or culturing the recombinant microorganisms describedherein, such that a desired compound (e.g., a desired unsaturated fattyacid) is produced. The term “culturing” includes maintaining and/orgrowing a living microorganism of the present invention (e.g.,maintaining and/or growing a culture or strain). In one embodiment, amicroorganism of the invention is cultured in liquid media. In anotherembodiment, a microorganism of the invention is cultured in solid mediaor semi-solid media. In a preferred embodiment, a microorganism of theinvention is cultured in media (e.g., a sterile, liquid media)comprising nutrients essential or beneficial to the maintenance and/orgrowth of the microorganism (e.g., carbon sources or carbon substrate,for example complex carbohydrates such as bean or grain meal, starches,sugars, sugar alcohols, hydrocarbons, oils, fats, fatty acids, organicacids and alcohols; nitrogen sources, for example, vegetable proteins,peptones, peptides and amino acids derived from grains, beans andtubers, proteins, peptides and amino acids derived form animal sourcessuch as meat, milk and animal byproducts such as peptones, meat extractsand casein hydrolysates; inorganic nitrogen sources such as urea,ammonium sulfate, ammonium chloride, ammonium nitrate and ammoniumphosphate; phosphorus sources, for example, phosphoric acid, sodium andpotassium salts thereof; trace elements, for example, magnesium, iron,manganese, calcium, copper, zinc, boron, molybdenum, and/or cobaltsalts; as well as growth factors such as amino acids, vitamins, growthpromoters and the like).

Preferably, microorganisms of the present invention are cultured undercontrolled pH. The term “controlled pH” includes any pH which results inproduction of the desired product (e.g., an unsaturated fatty acid). Inone embodiment, microorganisms are cultured at a pH of about 7. Inanother embodiment, microorganisms are cultured at a pH of between 6.0and 8.5. The desired pH may be maintained by any number of methods knownto those skilled in the art.

Also preferably, microorganisms of the present invention are culturedunder controlled aeration. The term “controlled aeration” includessufficient aeration (e.g., oxygen) to result in production of thedesired product (e.g., an unsaturated fatty acid). In one embodiment,aeration is controlled by regulating oxygen levels in the culture, forexample, by regulating the amount of oxygen dissolved in culture media.Preferably, aeration of the culture is controlled by agitating theculture. Agitation may be provided by a propeller or similar mechanicalagitation equipment, by revolving or shaking the growth vessel (e.g.,fermentor) or by various pumping equipment. Aeration may be furthercontrolled by the passage of sterile air or oxygen through the medium(e.g., through the fermentation mixture). Also preferably,microorganisms of the present invention are cultured without excessfoaming (e.g., via addition of antifoaming agents).

Moreover, plants or microorganisms of the present invention can becultured under controlled temperatures. The term “controlledtemperature” includes any temperature which results in production of thedesired product (e.g., an unsaturated fatty acid). In one embodiment,controlled temperatures include temperatures between 15° C. and 95° C.In another embodiment, controlled temperatures include temperaturesbetween 15° C. and 70° C. Preferred temperatures are between 20° C. and55° C., more preferably between 30° C. and 45° C. or between 30° C. and50° C.

Microorganisms can be cultured (e.g., maintained and/or grown) in liquidmedia and preferably are cultured, either continuously orintermittently, by conventional culturing methods such as standingculture, test tube culture, shaking culture (e.g., rotary shakingculture, shake flask culture, etc.), aeration spinner culture, orfermentation. In a preferred embodiment, the microorganisms are culturedin shake flasks. In a more preferred embodiment, the microorganisms arecultured in a fermentor (e.g., a fermentation process). Fermentationprocesses of the present invention include, but are not limited to,batch, fed-batch and continuous methods of fermentation. The phrase“batch process” or “batch fermentation” refers to a closed system inwhich the composition of media, nutrients, supplemental additives andthe like is set at the beginning of the fermentation and not subject toalteration during the fermentation, however, attempts may be made tocontrol such factors as pH and oxygen concentration to prevent excessmedia acidification and/or microorganism death. The phrase “fed-batchprocess” or “fed-batch” fermentation refers to a batch fermentation withthe exception that one or more substrates or supplements are added(e.g., added in increments or continuously) as the fermentationprogresses. The phrase “continuous process” or “continuous fermentation”refers to a system in which a defined fermentation media is addedcontinuously to a fermentor and an equal amount of used or “conditioned”media is simultaneously removed, preferably for recovery of the desiredproduct (e.g., an unsaturated fatty acid). A variety of such processeshave been developed and are well-known in the art.

The phrase “culturing under conditions such that a desired compound(e.g., an unsaturated fatty acid, for example, DHA) is produced”includes maintaining and/or growing plants or microorganisms underconditions (e.g., temperature, pressure, pH, duration, etc.) appropriateor sufficient to obtain production of the desired compound or to obtaindesired yields of the particular compound being produced. For example,culturing is continued for a time sufficient to produce the desiredamount of a unsaturated fatty acid (e.g., DHA). Preferably, culturing iscontinued for a time sufficient to substantially reach maximalproduction of the unsaturated fatty acid. In one embodiment, culturingis continued for about 12 to 24 hours. In another embodiment, culturingis continued for about 24 to 36 hours, 36 to 48 hours, 48 to 72 hours,72 to 96 hours, 96 to 120 hours, 120 to 144 hours, or greater than 144hours. In another embodiment, culturing is continued for a timesufficient to reach production yields of unsaturated fatty acids, forexample, cells are cultured such that at least about 15 to 20 g/L ofunsaturated fatty acids are produced, at least about 20 to 25 g/Lunsaturated fatty acids are produced, at least about 25 to 30 g/Lunsaturated fatty acids are produced, at least about 30 to 35 g/Lunsaturated fatty acids are produced, at least about 35 to 40 g/Lunsaturated fatty acids are produced (e.g., at least about 37 g/Lunsaturated fatty acids) or at least about 40 to 50 g/L unsaturatedfatty acids are produced. In yet another embodiment, microorganisms arecultured under conditions such that a preferred yield of unsaturatedfatty acids, for example, a yield within a range set forth above, isproduced in about 24 hours, in about 36 hours, in about 48 hours, inabout 72 hours, or in about 96 hours.

In producing unsaturated fatty acids, it may further be desirable toculture cells of the present invention in the presence of supplementalfatty acid biosynthetic substrates. The term “supplemental fatty acidbiosynthetic substrate” includes an agent or compound which, whenbrought into contact with a cell or included in the culture medium of acell, serves to enhance or increase unsaturated fatty acid biosynthesis.Supplemental fatty acid biosynthetic substrates of the present inventioncan be added in the form of a concentrated solution or suspension (e.g.,in a suitable solvent such as water or buffer) or in the form of a solid(e.g., in the form of a powder). Moreover, supplemental fatty acidbiosynthetic substrates of the present invention can be added as asingle aliquot, continuously or intermittently over a given period oftime.

The methodology of the present invention can further include a step ofrecovering a desired compound (e.g., an unsaturated fatty acid). Theterm “recovering” a desired compound includes extracting, harvesting,isolating or purifying the compound from culture media. Recovering thecompound can be performed according to any conventional isolation orpurification methodology known in the art including, but not limited to,treatment with a conventional resin (e.g., anion or cation exchangeresin, non-ionic adsorption resin, etc.), treatment with a conventionaladsorbent (e.g., activated charcoal, silicic acid, silica gel,cellulose, alumina, etc.), alteration of pH, solvent extraction (e.g.,with a conventional solvent such as an alcohol, ethyl acetate, hexaneand the like), dialysis, filtration, concentration, crystallization,recrystallization, pH adjustment, lyophilization and the like. Forexample, a compound can be recovered from culture media by firstremoving the microorganisms from the culture. Media is then passedthrough or over a cation exchange resin to remove unwanted cations andthen through or over an anion exchange resin to remove unwantedinorganic anions and organic acids having stronger acidities than theunsaturated fatty acid of interest (e.g., DHA).

Preferably, a desired compound of the present invention is “extracted,”“isolated” or “purified” such that the resulting preparation issubstantially free of other components (e.g., free of media componentsand/or fermentation byproducts). The language “substantially free ofother components” includes preparations of desired compound in which thecompound is separated (e.g., purified or partially purified) from mediacomponents or fermentation byproducts of the culture from which it isproduced. In one embodiment, the preparation has greater than about 80%(by dry weight) of the desired compound (e.g., less than about 20% ofother media components or fermentation byproducts), more preferablygreater than about 90% of the desired compound (e.g., less than about10% of other media components or fermentation byproducts), still morepreferably greater than about 95% of the desired compound (e.g., lessthan about 5% of other media components or fermentation byproducts), andmost preferably greater than about 98-99% desired compound (e.g., lessthan about 1-2% other media components or fermentation byproducts). Whenthe desired compound is an unsaturated fatty acid that has beenderivatized to a salt, the compound is preferably further free (e.g.,substantially free) of chemical contaminants associated with theformation of the salt. When the desired compound is an unsaturated fattyacid that has been derivatized to an alcohol, the compound is preferablyfurther free (e.g., substantially free) of chemical contaminantsassociated with the formation of the alcohol.

In an alternative embodiment, the desired unsaturated fatty acid is notpurified from the plant or microorganism, for example, when the plant ormicroorganism is biologically non-hazardous (e.g., safe). For example,the entire plant or culture (or culture supernatant) can be used as asource of product (e.g., crude product). In one embodiment, the plant orculture (or culture supernatant) supernatant is used withoutmodification. In another embodiment, the plant or culture (or culturesupernatant) is concentrated. In yet another embodiment, the plant orculture (or culture supernatant) is pulverized, dried, or lyophilized.

B. High Yield Production Methodologies

A particularly preferred embodiment of the present invention is a highyield production method for producing unsaturated fatty acids, e.g.,DHA, comprising culturing a manipulated plant or microorganism underconditions such that the unsaturated fatty acid is produced at asignificantly high yield. The phrase “high yield production method,” forexample, a high yield production method for producing a desired compound(e.g., for producing an unsaturated fatty acid) includes a method thatresults in production of the desired compound at a level which iselevated or above what is usual for comparable production methods.Preferably, a high yield production method results in production of thedesired compound at a significantly high yield. The phrase“significantly high yield” includes a level of production or yield whichis sufficiently elevated or above what is usual for comparableproduction methods, for example, which is elevated to a level sufficientfor commercial production of the desired product (e.g., production ofthe product at a commercially feasible cost). In one embodiment, theinvention features a high yield production method of producingunsaturated fatty acids that includes culturing a manipulated plant ormicroorganism under conditions such that an unsaturated fatty acid isproduced at a level greater than 2 g/L. In another embodiment, theinvention features a high yield production method of producingunsaturated fatty acids that includes culturing a manipulated plant ormicroorganism under conditions such that an unsaturated fatty acid isproduced at a level greater than 10 g/L. In another embodiment, theinvention features a high yield production method of producingunsaturated fatty acids that includes culturing a manipulated plant ormicroorganism under conditions such that an unsaturated fatty acid isproduced at a level greater than 20 g/L. In yet another embodiment, theinvention features a high yield production method of producingunsaturated fatty acids that includes culturing a manipulated plant ormicroorganism under conditions such that an unsaturated fatty acid isproduced at a level greater than 30 g/L. In yet another embodiment, theinvention features a high yield production method of producingunsaturated fatty acids that includes culturing a manipulated plant ormicroorganism under conditions such that an unsaturated fatty acid isproduced at a level greater than 40 g/L.

The invention further features a high yield production method forproducing a desired compound (e.g., for producing an unsaturated fattyacid) that involves culturing a manipulated plant or microorganism underconditions such that a sufficiently elevated level of compound isproduced within a commercially desirable period of time. In an exemplaryembodiment, the invention features a high yield production method ofproducing unsaturated fatty acids that includes culturing a manipulatedplant or microorganism under conditions such that an unsaturated fattyacid is produced at a level greater than 15-20 g/L in 36 hours. Inanother embodiment, the invention features a high yield productionmethod of producing unsaturated fatty acids that includes culturing amanipulated plant or microorganism under conditions such that anunsaturated fatty acids produced at a level greater than 25-30 g/L in 48hours. In another embodiment, the invention features a high yieldproduction method of producing unsaturated fatty acids that includesculturing a manipulated plant or microorganism under conditions suchthat an unsaturated fatty acids produced at a level greater than 35-40g/L in 72 hours, for example, greater that 37 g/L in 72 hours. Inanother embodiment, the invention features a high yield productionmethod of producing unsaturated fatty acids that includes culturing amanipulated plant or microorganism under conditions such that anunsaturated fatty acid is produced at a level greater than 30-40 g/L in60 hours, for example, greater that 30, 35 or 40 g/L in 60 hours. Valuesand ranges included and/or intermediate within the ranges set forthherein are also intended to be within the scope of the presentinvention. For example, unsaturated fatty acid production at levels ofat least 31, 32, 33, 34, 35, 36, 37, 38 and 39 g/L in 60 hours areintended to be included within the range of 30-40 g/L in 60 hours. Inanother example, ranges of 30-35 g/L or 35-40 g/L are intended to beincluded within the range of 30-40 g/L in 60 hours. Moreover, theskilled artisan will appreciate that culturing a manipulatedmicroorganism to achieve a production level of, for example, “30-40 g/Lin 60 hours” includes culturing the microorganism for additional timeperiods (e.g., time periods longer than 60 hours), optionally resultingin even higher yields of an unsaturated fatty acid being produced.

IV. Compositions

The desaturase nucleic acid molecules, proteins, and fragments thereof,of the invention can be used to produce unsaturated fatty acids whichcan be incorporated into compositions. Compositions of the presentinvention include, e.g., compositions for use as animal feed,compositions for use as nutraceuticals (e.g., dietary supplements), andpharmaceutical compositions suitable for administration. Suchpharmaceutical compositions typically comprise an unsaturated fatty acidand a pharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a LCPUFA, or a fragment thereof, produced by the nucleicacid and protein molecules of the present invention) in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle which contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying which yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

As defined herein, a therapeutically effective amount of protein orpolypeptide (i.e., an effective dosage) ranges from about 0.001 to 30mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, morepreferably about 0.1 to 20 mg/kg body weight, and even more preferablyabout 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6mg/kg body weight. The skilled artisan will appreciate that certainfactors may influence the dosage required to effectively treat asubject, including but not limited to the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject, and other diseases present. Moreover, treatment of a subjectwith a therapeutically effective amount of a protein, polypeptide, orantibody can include a single treatment or, preferably, can include aseries of treatments.

In a preferred example, a subject is treated with a LCPUFA in the rangeof between about 0.1 to 20 mg/kg body weight, one time per week forbetween about 1 to 10 weeks, preferably between 2 to 8 weeks, morepreferably between about 3 to 7 weeks, and even more preferably forabout 4, 5, or 6 weeks. It will also be appreciated that the effectivedosage of antibody, protein, or polypeptide used for treatment mayincrease or decrease over the course of a particular treatment. Changesin dosage may result and become apparent from the results of diagnosticassays as described herein.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication, as well as the figures, are incorporated herein byreference.

EXAMPLES

Materials:

Thraustochytrium s.p ATCC 21685 and Pythium irregulare were purchasedfrom American type culture collection (12301 Parklawn Drive, Rockville,Md., 20852 USA) and grown in a medium (Weete, J. D., et al. (1997)Lipids 32:839-845) at 24° C. for 7 days. After then biomass wereharvested by centrifugation and used for RNA isolation.

Example 1: Construction and Screening of cDNA Library

Total RNA was isolated from the above materials according to Qiu andErickson (Qiu, X. and Eriekson, L. (1994) Plant Mol. Biol. Repr.12:209-214). The cDNA library was constructed from the total RNA. Thefirst strand cDNA was synthesized by superscript II reversetranscriptase from Gibco-BRL. The second strand cDNA was synthesized byDNA polymerase I from Stratagene. After size fractionation, cDNA insertslarger than 1 kb were ligated into λ Uni-Zap XR vector (Stratagene). Therecombinant DNAs were then packed with Gigapack III Gold packagingextract (Stratagene) and plated on NZY plates. The resulting libraryrepresented more than 5×106 independent clones. Screening of the cDNAlibrary was performed according to standard methods (Sambrook, J,Fritseh, E. F., Maniatis, T. (1989) Molecular cloning—A laboratorymanual. (Cold Spring Harbor, N.Y., USA.)

Example 2: RT-PCR

The single strand cDNA was synthesized by superscript II reversetranscriptase (Gibco-BRL) from total RNA and was then used as thetemplate for PCR reaction with two degenerate primers (The forwardprimer: GCNCA/GANGANCAC/TCCNGGXGG (SEQ ID NO:9) and the reverse primer:ATNTG/TNGGA/GAANAG/AG/ATGG/ATG (SEQ ID NO:10)). The PCR amplificationconsisted of 35 cycles with 1 min at 94° C., 1.5 min at 55° C. and 2 minat 72° C. followed by an extension step at 72° C. for 10 min. Theamplified products from 800 bp to 1000 bp were isolated from agarose geland purified by a kit (Qiaex II gel purification, Qiagen), andsubsequently cloned into the TA cloning vector pCR®2.1 (Invitrogen). Thecloned inserts were then sequenced by PRISM DyeDeoxy Terminator CycleSequencing System (Perkin Elmer/Applied Biosystems).

Example 3: Expression of Fad4, Fad5, Fad5-2, and Fad6 in Yeast

The open reading frames of Fad4, Fad5, Fad5-2, and Fad6 were amplifiedby PCR using the Precision Plus enzyme (Stratagene) and cloned into a TAcloning vector (pCR®2.1, Invitrogen). Having confirmed that the PCRproducts were identical to the original cDNAs by sequencing, thefragments were then released by a BarnHI-EcoRI double digestion andinserted into the yeast expression vector pYES2 (Invitrogen) under thecontrol of the inducible promoter GALL

Yeast strains InvSc2 (Invitrogen) was transformed with the expressionconstructs using the lithium acetate method and transformants wereselected on minimal medium plates lacking uracil (Gietz, D., et al.(1992) Nucleic Acids Res. 20:1425; Covello, P. S. and Reed, D. W. (1996)Plant Physiol. 111:223-226).

Transformants were first grown in minimal medium lacking uracil andcontaining glucose at 28° C. After overnight culture, the cells werespun down, washed and resuspended in distilled water. Minimal mediumcontaining 2% galactose, with or without 0.3 mM substrate fatty acids inthe presence of 0.1% tergitol, was inoculated with the yeasttransformant cell suspension and incubated at 20° C. for three days, andthen 15° C. for another three days.

Example 4: Fatty Acid Analysis

Thraustochytrium, Pythium irregulare and yeast cells were harvested andwashed twice with distilled water. Then 2 mL methanolic KOH (7.5% w/vKOH in 95% methanol) was added to the materials and the mixture sealedin a 12 ml glass culture tube was heated to 80° C. for 2 hours. 0.5 mLwater was added and the sample was extracted twice with 2 mL hexane toremove the non-saponifiable lipids. The remaining aqueous phase was thenacidified by adding 1 mL 6 N HCl and extracted twice with 2 mL hexane.The hexane phases were combined and dried under a stream of nitrogen. 2mL 3 N methanolic HCl (SUPELCO, Supelco Park, Bellefonte, Pa.16823-0048) was added and the mixture was heated at 80° C. for 2 hours.After cooling to room temperature, 1 mL 0.9% NaCl was added and themixture extracted twice with 2×2 mL hexane. The combined hexane wasevaporated under nitrogen. The resulting fatty acid methyl esters(FAMEs) were analyzed by GC and GC-MS according to Covello & Reed(Covello, P. S. and Reed, D. W. (1996) Plant Physiol. 111:223-226).

GC/MS analysis was performed in standard EI mode using a Fisons VG TRIO2000 mass spectrometer (VG Analytical, UK) controlled by Masslynxversion 2.0 software, coupled to a GC 8000 Series gas chromatograph. ADB-23 column (30M×0.25 mm i.d., 0.25 Ilm film thickness, J&W Scientific,Folsom, Calif.) that was temperature-programmed at 180° C. for 1 min,then 4 C/rnin to 240° C. and held for 15 minutes, was used for FAMEanalysis.

Example 5: Transformation of Brassica juncea and Flax (Linumusitatissimum) and Exogenous Fatty Acid Treatment

The hypocotyls of 5-6 day seedlings of B. juncea and flax were used asexplants for inoculation with the Agrobacterium tumefaciens that hostsbinary vectors with the full-length cDNAs under the control of thedifferent promoters. The 20-day transgenic seedlings were used forexogenous fatty acid treatment. The seedling was divided into threeparts: leaves, stems and roots. Each was cut into the small pieces andplaced in a 24-well titer plate. To each well, 2 mL 0.05% sodium salt ofsubstrates (NuCheck Prep Inc., Elysian, Minn.) was added. The plate wasthen incubated at 24° C. for 4 h with gentle shaking. After incubation,the plant tissues were washed three times with water and then used forfatty acid analysis.

Example 6: Fatty Acid Profile of the Thrauschytrium Sp.

Thraustochytrium and Pythium irregulare have recently drawn scientificattention due to its ability in production of LCPUFAs such as DHA, AA,EPA and DPA. FIGS. 23 and 24 show the fatty acid composition of thelipids isolated from 7 day cultures of Thraustochytrium sp. and Pythiumirregulare, respectively. As shown in the tables, the microorganismscontain a broad range of polyunsaturated fatty acids, from both n−3 andn−6 families, from 1 8-carbon A6 fatty acids (gamma-linolenic acid andsteardonic acid) to 22-carbon Δ4 fatty acids (DHA and DPA). Theorganisms, especially Thraustochytrium sp., appear to contain a full-setof desaturation and elongation enzymes required for the DHA and DPAbiosynthesis. The strain lacks 24-carbon polyunsaturated fatty acids,the proposed precursors for DHA and DPA synthesis in Precher's pathway(Voss, A., et al. (1991) J. Biol. Chem. 266:19995-20000; Mohammed, B.S., et al. (1997) Biochem. J. 326:425-430). The 24-carbon fatty acid maynot be involved in in vivo synthesis of 22-carbon Δ4 fatty acids such asDHA and DPA in Thraustochytrium sp.

Example 7: Identification of cDNAs Coding for the “Front-End” Desaturase

To identify genes coding for desaturases involved in biosynthesis ofLCFUFAs in Thraustochytrium sp. and Pythium irregulare, a PCR-basedcloning strategy was adopted. Two degenerate primers are designed totarget the heme-binding motif of N-terminal extension of cyt b5-likedomain in front-end desaturases and the third conservative histidinemotif in all microsomal desaturases, respectively. The rational behindthe design is that the desaturases involved in EPA and DHA biosynthesisin Thraustochytrium sp. and Pythium irregulare, should have similarprimary structure as other front-end desaturases, i.e. N-terminalextension of cyt b5-like domain in the desaturase. Four cDNAs fragmentswere identified from Thraustochytrium sp. and Pythium irregulare thatencode fusion proteins containing cyt b5-like domain in the N-terminus.

To isolate full-length cDNA clones, the four inserts were used as probesto screen cDNA libraries of Thraustochytrium sp. and Pythium irregulare,which resulted in identification of several cDNA clones in each group.Sequencing of all those clones identified four full-length cDNAs whichwere named as Fad4, Fad5, Fad5-2 and Fad6. The open reading frame ofFad4 is 1560 bp and codes for 519 amino acids with molecular weight of59.1 kDa (FIG. 1). Fad5 is 1230 bp in length and codes for 439 aminoacids with molecular weight of 49.8 kDa (FIG. 2). A sequence comparisonof these two sequences from Thraustochytrium sp. showed only 16% aminoacid identity between the deduced proteins. A detailed analysis revealedthat Fad4 is 80 amino acids longer than Fad5, occurring between thesecond and third conservative histidine motifs (FIG. 3). The openreading frame of Fad5-2 from Pythium irregulare is 1371 bp and codes for456 amino acids (FIG. 4). Fad6 from Pythium irregulare is 1383 bp inlength and codes for 460 amino acids (FIG. 5). Sequence comparison ofthe two sequences from Pythium irregulare showed over 39% similaritybetween the deduced proteins (FIG. 6).

A BLASTP™ search of the protein database revealed the following hits foreach of the four proteins, Fad4, Fad5, Fad5-2, and Fad6:

Fad 4 (519 amino acid residues) Blastp nr Accession No. OrganismDescription Length % Identity AF067654 Mortierella Δ5 fatty acid 509 29alpina desaturase AF054824 Mortierella Δ5 microsomal 509 28 alpinadesaturase AB022097 Dictyostelium Δ5 fatty acid 507 27 discoideumdesaturase AB029311 Dictyostelium fatty acid 519 26 discoideumdesaturase L11421 Synechocystis sp. Δ6 desaturase 410 25 D90914

Fad 5 (439 amino acid residues) Blastp nr Accession No. OrganismDescription Length % Identity AF139720 Euglena gracilis Δ8 fatty acid404 29 desaturase AF007561 Borago officinalis Δ6 desaturase 421 27U79010 Borago officinalis Δ6 desaturase 421 27 AF309556 Danio rerio Δ6fatty acid 422 26 desaturase AF110510 Mortierella Δ6 fatty acid 463 25alpina desaturase

Fad 5-2 (456 amino acid residues) Blastp nr Accession No. OrganismDescription Length % Identity AB029311 Dictostelium Fatty acid 443 41discoideum desaturase AB022097 Dictostelium Δ5 fatty acid 445 39discoideum desaturase AF067654 Mortierella Δ5 fatty acid 441 38 alpinadesaturase AF054824 Mortierella Δ5 microsomal 441 38 alpina desaturaseL11421 Synechocystis sp. Δ6 desaturase 361 28 D90914

Fad 6 (459 amino acid residues) Blastp nr Accession No. OrganismDescription Length % Identity AF110510 Mortierella Δ6 fatty acid 437 38alpina desaturase AB020032 Mortierella Δ6 fatty acid 437 38 alpinadesaturase AF306634 Mortierella Δ6 fatty acid 437 38 isabellinadesaturase AF307940 Mortierella Δ6 fatty acid 438 38 alpina desaturaseAJ250735 Ceratodon Δ6 fatty acid 438 36 purpureus desaturase

Example 8: Expression of Fad4, Fad5, Fad5-2, and Fade in Yeast

To confirm the function of Fad4, the full-length cDNA was expressed inthe yeast strain InvSc2 under the control of the inducible promoter.FIG. 7 shows that with supplementation of the medium with 22:5(7,10,13,16,19), yeast cells containing Fad4 cDNA had an extra fattyacid as compared to the vector control. The peak has a retention timeidentical to the DHA standard. LC/MS analysis of the free fatty acidshowed that it yields deprotonated molecular ions (m/z=279) identical tothe DHA standard in negative ion electrospray. Moreover, GC/MS analysisof the FAME confirmed that the spectrum of the peak is identical to thatof the DHA standard (FIGS. 8A and 8B). These results indicate that Fad4is a Δ4 fatty acid desaturase which is able to introduce a double bondat position 4 of the 22:5(7,10,13,16,19) substrate, resulting in a Δ4desaturated fatty acid, DHA (22:6-4,7,10,13,16,19).

To further study the substrate specificity of the Fad4, a number ofsubstrates including 18:2(9,12), 18:3(9,12,15), 20:3(8,11,14) and22:4(7,10,13,16) were separately supplied to the yeast transformants.The results indicated Fad4 could also use 22:4 (7,10,13,16) as asubstrate (FIG. 9) to produce another Δ4 desaturated fatty acid, DPA(22:5-4,7,10,13,16) (FIGS. 10A and 10B). The rest of the fatty acidsexamined were not effective substrates.

To confirm the function of Fad5 and Fad5-2, the S. cerevisiae Invsc2 wastransformed with plasmids, which contain the open reading frame of theFad5 and Fad5-2 respectively under the control of thegalactose-inducible promoter. When the yeast transformants were inducedby galactose in a medium containing homo-gamma-linolenic acid (HGLA,20:3-8,11,14), an extra peak was observed in the chromatogram of FAMEsaccumulating in the transformants compared with the control (FIG. 11). Acomparison of the chromatogram with that of the standards revealed thatthe fatty acid had a retention time identical to the arachidonic acidstandard (AA, 20:4-5,8,11,14). To further confirm the regiochemistry ofthe products, the FAMEs were analyzed by GC/MS. FIGS. 12A and 12Bindicate that the mass spectra of the new fatty acid and the AA standardare identical. These results demonstrate that Fad5 and Fad5-2 convertHGLA (20:3-8,11,13) into AA (20:4-5,8,11,14) in yeast.

To further study the substrate specificity of Fad5-2, the plasmidcontaining Fad5-2 was transferred into another yeast strain AMY-2α whereole1, a Δ9 desaturase gene, is disrupted. The strain is unable to growin minimal media without supplementation with mono-unsaturated fattyacids. In this experiment, the strain was grown in minimal mediumsupplemented with 17:1(10Z), a non-substrate of Fad5-2, which enabledstudy of the specificity of the enzyme towards various substrates,especially monounsaturates. A number of possible substrates including16:1(94, 18:1(94, 18:1(114, 18:1(11E), 18:1(12E), 18:1(154,18:2(9Z,12Z), 18:3(9Z,12Z,15Z), 20:2(11Z,14Z) and 20:3(11Z,14Z,17Z) weretested. Results indicated that Fad5-2 could desaturate unsaturated fattyacids with Δ9 ethylenic and Δ11 ethylenic double bonds, as well as thefatty acid with Δ8 ethylenic double bond (20:3-8,11,14). As shown inFIG. 13, Fad5-2 effectively converted both 18:1(9Z) and 18:1(11Z)substrates into their corresponding Δ5 desaturated fatty acids, 18:2-5,9(the retention time 10.34 min) and 18:1-5,11 (the retention time 10.44min), respectively. Fad5-2 also desaturated trans fatty acid such as18:1(11E) and 18:1(12E).

FIG. 25 is a comparison of substrate preference of Fad5-2 for fatty acidsubstrates tested in the yeast strain AMY-2α. The relative proportionsof the substrates and the products accumulated are a useful indicator ofsubstrate preference of the enzyme. As shown in FIG. 25, Fad5-2 prefersfatty acids with 20-carbon as substrates, such as 20:3(8Z,11Z,14Z),20:3(11Z,14Z,17Z) and 20:2(11Z,14Z). Whereas, the shorter chain fattyacid is a relatively weaker substrate for the enzyme in yeast.

To confirm the function of Fad6, the S. cerevisiae host strain Invsc2was transformed with a plasmid containing the open reading frame of Fad6under the control of the galactose-inducible promoter, GALL When theyeast transformant was induced by galactose in a medium containinglinoleic acid, an extra peak was observed in the chromatogram of theFAMEs accumulating in the transformants compared with the control (FIG.14). A comparison of the chromatogram with that of the standardsrevealed that the peak had a retention time identical to thegamma-linolenic acid (GLA, 18:3-6,9,12) standard. To confirm theregioselectivity of the products, the diethylamine derivatives of fattyacids from the expressing strain were analyzed by GC-MS. FIG. 15 showsthat the new peak is indeed GLA with three double bonds at the Δ6, Δ9,and Δ12 positions. Major fragments of n and n+1 carbons differing by 12D are diagnostic of a double bond between carbon n+1 and n+2. Thus, thefragments at 156 and 168, 196 and 208, and 236 and 248, indicate doublebonds at the Δ6, Δ9, and Δ12 positions, respectively. These resultsdemonstrate that Fad6 is a Δ6 desaturase that converts linoleic acid(18:2) to GLA in yeast.

Example 9: Expression of Fad4 in B. juncea

To determine whether Traustochytrium Fad4 is functional in oilseedcrops, B. juncea were transformed with the construct containing Fad4under the control of a constitutive promoter. Eight independenttransgenic plants were obtained. In B. juncea there is no Δ4 fatty aciddesaturase substrates available. Thus, to examine the activity of thetransgenic enzyme in the plants, the 22:5 (n−3) substrate must beexogenously supplied. In this experiment, both wild type and transgenicswere applied with an aqueous solution of sodium docosapentaenoate. Itwas found that exogenously applied substrates were readily taken up byroots, stem, and leaves of both types of plants, but converted into DHAonly in transgenics. Leaves have a higher level of production of DHAthan roots and stems. In leaves, the exogenous substrate wasincorporated to a level of 10-20% of the total fatty acids and Δ4desaturated fatty acid (22:6, n−3) was produced in a range of 3-6% ofthe total fatty acids (FIG. 16). These results indicate that the Δ4fatty acid desaturase from Traustochytrium is functional in oilseedcrops.

Example 10: Expression of Fad5-2 in B. juncea

To determine whether Fad5-2 is functional in oilseed crops and itsexpression has any effect on their growth and development, B. junceawere transformed with a binary vector that contained Fad5-2 cDNA behinda constitutive promoter (a tandem cauliflower mosaic virus 35Spromoter). Six independent primary transgenic plants were obtained andthe fatty acid profile of lipids from different tissues was determined.FIG. 17 shows the fatty acid composition of three-week-old seedlingplants from one T₁ line. Compared with wild type, all transgenic planttissues have an altered fatty acid profile containing four additionalpeaks which were identified as four different Δ5-undesaturatedpolymethylene-interrupted fatty acids (Δ5-UPIFAs), specifically,taxoleic (18:2-5,9); ephedrenic (18:2-5,11); pinolenic (18:3-5,9,12),and coniferonic acids (18:4-5,9,12,15). Thus B. juncea, like yeast, canfunctionally express the P. irregulare Δ5 desaturase to convert theendogenous substrates 18:1-9; 18:1-11; 18:2-9,12, and 18:3-9,12,15 tothe corresponding Δ5 desaturated fatty acids. The roots produced thehighest amount of the Δ5-UPIFAs, representing more than 20% of the totalfatty acids, followed by 6% in stems and 5% in leaves (FIG. 17).

In B. juncea there is no homo-gamma-linolenic acid (20:3-8,11,14)substrate available. Thus, to examine whether the transgenic plant canproduce AA, the substrate 20:3(8,11,14) was exogenously supplied. Inthis experiment, both wild type and transgenics were applied with anaqueous solution of sodium homo-gamma-linolenate. It was found thatexogenously applied substrates were readily taken up by roots, stem, andleaves of transgenic plants and converted into AA in plants (FIG. 18).

There was no observable phenotypic effect on the growth and developmentin the transgenic B. juncea, although the Δ5-UPIFAs accumulated in allparts of the plant. Growth and differentiation of vegetative tissuessuch as the leaves, stems, and roots were indistinguishable from thecorresponding wild type.

To produce Δ5 desaturated fatty acids in seeds, B. juncea weretransformed with the construct containing Fad5-2 cDNA behind aheterologous seed-specific promoter (B. napus napin promoter). Fattyacid analysis of transgenic seeds showed that there were two new fattyacids appearing in the gas chromatogram of transgenics compared with thewild type control (FIG. 19). They were identified as taxoleic acid(18:2-5,9) and pinolenic acids (18:3-5,9,12). Together, these fattyacids represent 9.4% of the seed fatty acids. Accumulation of Δ5-UPIFAshas no significant effect on the oleic acid content compared with theuntransformed control.

Example 11: Expression of Fad5-2 in Flax

To produce Δ5 desaturated fatty acids in flax seeds, flax wastransformed with Fad5-2 under the control of two seed-specificpromoters, a heterologous B. napus napin promoter, and a flax endogenouspromoter. As shown in FIG. 26, transgenic plants containing the napinpromoter produced one Δ5 desaturated fatty acid, taxoleic acid in seedsat the level of less than 1% of the total fatty acids. Whereastransgenic plants containing the flax seed-specific promoter producedthree Δ5 desaturated fatty acids: taxoleic, pinolenic, and coniferonicacid. Of these, taxoleic (18:2-5,9) was the most abundant and accountedfor more than 9% of the total fatty acids in a elite line (FN-10-1),followed by coniferonic and pinolenic acids. Surprisingly, accumulationof Δ5 desaturated fatty acids in transgenic seeds has significant impacton the composition of other fatty acids, especially the oleic acidlevel. Accumulation of Δ5-UPIFAs was accompanied by a huge increase ofthe oleic acids in both types of transgenic plants expressing Fad5-2desaturase under the control of the different promoters. The content ofoleic acid in transgenic plants with the napin and flax seed-specificpromoters, on the average, reached 44.7% and 24.3% of the total fattyacids, respectively, relative to the untransformed control at 17.4%.

Example 12: Expression of Fad6 in Flax

To produce Δ6 desaturated fatty acids in flax seeds, two types of flaxwere transformed with the construct that contains Fad6 cDNA under thecontrol of a heterologous seed-specific promoter (B. napus napinpromoter). Type I flax (Normandy) is a traditional industrial oilseedcrop, whereas Type II (Solin) is an edible oilseed crop derived fromchemical mutagenesis of Type I. A total of twelve transgenic plants wereproduced. The majority of transgenics exhibited two novel fatty acidswhose retention times correspond to GLA and SDA and they constitute 0.1to 4.3% of the total fatty acids (FIG. 27). The level of GLA intransgenic Solin type is higher than that of SDA, while GLA intransgenic Normandy is lower than SDA. This is understandable becauselinoleic acid is a major fatty acid in Solin type linseed whileα-linolenic acid is a major fatty acid in Normandy seeds.

Example 13: Expression of Fad6 in B. juncea

To produce Δ6 desaturated fatty acids in seeds of B. juncea, B. junceawere transformed with the same construct used in flax transformation,i.e., Fad6 under the control of the B. napus napin promoter. Fifteenindependent transgenic plants were obtained. Fatty acid analysis of thetransgenic seeds showed that there were three new fatty acids in the gaschromatogram of most transgenics compared with the wild type control(FIG. 20). The three fatty acids were identified as 18:2(6,9) and18:3(6,9,12), and 18:4(6,9,12,15). B. juncea, like flax, can alsofunctionally express Fad6 from P. irregulare, introducing a double bondat position 6 of endogenous substrate 18:1(9), 18:2(9,12), and18:3(6,9,12) resulting in production of three corresponding Δ6 fattyacids in the transgenic seeds. Among the three new fatty acids producedin transgenic seeds, GLA is the most abundant one, with a level intransgenic seeds of 30% to 38% of the total fatty acids. The next mostabundant component is SDA, which accounts for 3-10% of the total fattyacids in several transgenic lines (FIG. 21).

The fatty acid compositions of transgenic seeds are shown in FIG. 22. Itis clear that the high level production of Δ6 desaturated fatty acids isat the cost of two major fatty acids, linoleic and linolenic acids.Proportions of oleic and stearic acids in transgenics are slightlyreduced, but not significantly compared to those in the wild typecontrol. The content of linoleic acid in the transgenics wasdramatically reduced. In the untransformed wild type, linoleic acidaccounts for more than 40% of the total fatty acids in seeds. Intransgenics, the level was reduced to less than 10%.

As compared to the reduction of linoleic acid in transgenics, thedecrease in linolenic acid in transgenics is less dramatic, but stillsignificant. In the untransformed wild type, linolenic acid accounts formore than 10% of the total fatty acids in seeds while in transgenics thelevel was reduced to less than 5%. The two dramatically reduced fattyacids in transgenic seeds are the substrates of the Δ6 desaturase, andthe reduction is the cost for producing two corresponding Δ6 desaturatedfatty acids.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

We claim:
 1. A method for producing n−3 long chain polyunsaturated fattyacids comprising at least 20 carbons in a transgenic plant, comprising:a) selecting a first plant line having high content of n−6 fatty acidlinoleic acid (LA) relative to a second plant line of the same plantspecies; b) providing at least one nucleic acid molecule encoding a Δ6desaturase in said first plant line to produce a transgenic plant line,wherein the level of n−6 gamma-linolenic acid (GLA) produced in saidfirst transgenic plant line is higher than the level of GLA produced ina reference transgenic plant line of the second plant line expressing anucleic acid molecule encoding the same Δ6 desaturase, c) furthermanipulating the first transgenic plant to comprise the desaturase andelongase activities for biosynthetic production of n−3 long chainpolyunsaturated fatty acids comprising at least 20 from GLA; d)cultivating the transgenic plant line under conditions to produce then−3 long chain polyunsaturated fatty acids comprising at least 20carbon; and e) optionally recovering the n−3 long chain polyunsaturatedfatty acids comprising at least 20 carbons.
 2. The method of claim 1,wherein the n−3 long chain polyunsaturated fatty acids comprises atleast one of eicosapentaenioc acid (EPA, 20:5) and docosahexaenoic acid(DHA, 22:6).
 3. The method of claim 1, wherein the n−3 long chainpolyunsaturated fatty acids comprises docosahexaenoic acid (DHA, 22:6).4. The method of claim 1, wherein said at least one Δ6 desaturasecomprises a cytochrome b5-like domain.
 5. The method of claim 4, whereinsaid at least one Δ6 desaturase further comprises at least two histidinemotifs.
 5. The method of claim 4, wherein said at least one Δ6desaturase comprises three histidine motifs.
 6. The method of claim 1,wherein said at least one Δ6 desaturase has at least 70% sequenceidentity to the amino acid sequence of SEQ ID NO:
 8. 7. The method ofclaim 1, wherein said at least one Δ6 desaturase comprises: a) the aminoacid sequence of SEQ ID NO: 8; or b) an amino acid sequence encoded bythe nucleotide sequence of SEQ ID NO:
 7. 8. The method of claim 1,wherein the plant species is a Brassica species.
 9. The method of claim3, wherein recovering the n−3 long chain polyunsaturated fatty acidscomprises isolating unsaturated fatty acids from said transgenic plantline.
 10. The method of claim 3, wherein d) cultivating the transgenicplant line comprises producing a transgenic plant to produce the n−3long chain polyunsaturated fatty acids comprising at least 20 carbons.11. The method of claim 10, wherein recovering the n−3 long chainpolyunsaturated fatty acids comprises isolating unsaturated fatty acidsfrom said transgenic plant.
 12. The method of claim 11, wherein saidunsaturated fatty acids are isolated from seeds of said transgenicplant.
 13. The method of claim 11, wherein said n−3 long chainpolyunsaturated fatty acids comprises DHA.
 14. The method of claim 9,wherein said unsaturated fatty acids are isolated in the form of lipids.15. The method of claim 9, wherein said n−3 long chain polyunsaturatedfatty acids comprises DHA.
 16. A composition comprising the unsaturatedfatty acids produced in claim
 11. 17. A dietary supplement, animal feed,a neutraceutical, or a pharmaceutical composition comprising theunsaturated fatty acids produced in claim 11.